21bb3034 Bijlage 1 behorende bij 21bb437 - Phoslock toxicity evaluation

PHOSLOCK® TOXICITY EVALUATION Kralingse Plas

-Final version-

• Authors: Karin Finsterle, Maíra Mucci, Yvon Verstijnen & Fons Smolders • Date: 4-11-2020 i

Titel/Title: Phoslock® Toxicity Evaluation, Kralingse Plas Status: Eindversie/Final version Auteurs/Authors: Karin Finsterle (Limnological Solutions) Maíra Mucci (Limnological Solutions) Yvon Verstijnen (Onderzoekcentrum B-WARE) Fons Smolders (Onderzoekcentrum B-WARE) Projectnummer/Project number: PR19-191A Rapportnummer/Report number: RP-19.191A.20.104 Opdrachtgever/Client: Gemeente Rotterdam

Informatie/Information: Onderzoekcentrum B-WARE BV Radboud Universiteit Nijmegen Mercator III, Toernooiveld 1 6525 ED Nijmegen, NL Limnological Solutions International Pty Ltd (LSI) Contact B-WARE Research Centre:

Contact LSI:

Yvon Verstijnen

Maíra Mucci

T: +31(0)24-2122206

T: +31(0)633364857

E: y.verstijnen@b-ware.eu

E: mm@limnosolutions.com

www.b-ware.eu

www.limnological-solutions.com

© Onderzoekcentrum B-WARE Nijmegen, 2020 ii

Summary Phoslock® In many freshwater lakes, eutrophication is an ongoing process. To reduce the availability of excess phosphorus (P) in the water column and restore water quality in such lakes, geo-engineering tools, such as Phoslock®, have been developed. Its development, appropriate use, and exact dose calculation are all based on solid scientific principles. Phoslock® contains 95 % bentonite clay and 5 % lanthanum. Lanthanum (La) is a naturally occurring element, also present in small quantities in European lake sediments. In Phoslock®, La is strongly associated with the bentonite clay but retains its capacity to bind phosphate. Once Phoslock® is applied to freshwaters, it disperses into small particles, binds P to form insoluble lanthanum-phosphate, and settles quickly on the lake bottom. It is not released as soluble La into the water. The safe use of Phoslock® is related to its active ingredient (La) and also to physical effects caused by a temporary increase in turbidity in the water during application. In this review, the evaluation of risks associated with the use of Phoslock® is therefore based on the (ecotoxicological) assessment of exposed organisms of the aquatic environment and exposed terrestrial mammals including humans. Ecotoxicological laboratory studies with aquatic organisms have been conducted with Phoslock® and soluble La-salts (e.g. LaCl3).

As soluble La-salts have different chemical properties to Phoslock®, ecotoxicological studies done with La-salts are not comparable to Phoslock® applications and, therefore, not suitable for the assessment of Phoslock® toxicity. Furthermore, these studies are often conducted under non-realistic conditions (such as the use of phosphate-free medium). Consequently, only ecotoxicological assays conducted with Phoslock® form the basis of this review. Toxicity and biodiversity effects During a Phoslock® application, the amount of suspended solids will increase in the short term. This temporary increase might have a short-term impact on organisms. Once Phoslock® is applied, however, it will quickly settle to the lake sediment, most of it within one day. Any potential undesired side effects can easily be prevented by dividing the application over several days. Once Phoslock® is applied it will strongly and irreversibly bind P, hence, there will be less P available for phytoplankton and cyanobacterial growth. Thus, any observed adverse effect on algal growth is expected to occur due to phosphate limitation rather than to direct toxic effects. Phytoplankton surveys after whole lake applications of Phoslock® have shown that there are no non-target impacts on phytoplankton due to the treatment.

The expected shift after treatment towards more mesotrophic conditions, with less algal biomass, involves an increase in the algal biodiversity: less cyanobacteria and higher diversity of green algae and diatoms. This desired post-treatment effect is commonly observed after a strong P reduction. Changes in the trophic state of a lake will also affect other communities in the food chain. For instance, one of the consequences of less phytoplankton is an increase in water transparency, which will positively affect macrophytes. Many field studies indeed show an increase in water transparency, followed by an increase in macrophytes after Phoslock® treatment (see figure below). A thin layer of Phoslock® on top of leaves after an application could have a potential impact on some macrophytes, however this is highly unlikely in the natural environment, where wind and waves will remove any Phoslock® from the leaves. Field studies have shown increased macrophyte coverage, accompanied by an increase in species number and colonization depths after Phoslock® treatment. Toxicity studies undertaken on zooplankton (e.g., Daphnia) have shown that Phoslock® has no toxic effect when used at recommended dosages and under realistic conditions. The results also show a wide margin of safety, related to field dosages.

Overall, field studies in which zooplankton surveys have been undertaken show a normal development of the zooplankton community after the application of Phoslock® (see figure below). iii

Likewise for tested macroinvertebrate species (e.g., amphipods and midge larvae) in toxicity studies, the application of realistic Phoslock® doses show a wide safety margin. Even at high test concentrations, no significant impacts have been observed in any of the tested species. This ensures that a considerable safety margin is maintained when Phoslock® is applied at the recommended dose rates. Several studies have been conducted to investigate the impact of Phoslock® on community development of benthic macroinvertebrates. Some studies found no differences in macroinvertebrate communities between pre- and post-treatment. Other studies found a (short-term) effect on some macroinvertebrate groups. Changes in the macroinvertebrate community, however, can be attributed to the change in trophic state of the lake, as increased macrophyte abundance leads to increased habitat complexity, promoting macrofauna.

See Figure 4, page 23: from left to right (a) untreated system with cyanobacterial bloom in spring/summer (b) system undergoing Phoslock® treatment in autumn/winter and (c) treated system in following spring/summer.

The toxicity studies conducted on fish also show a wide margin of safety (at realistic Phoslock® doses) in the case of sentinel fish species. Based on these data, no risks to fish are expected with applications of Phoslock® at standard dose rates. The reported and expected changes in increased macrophyte coverage in the field and subsequent macroinvertebrate diversity can positively influence the fish stock, due to increased diversity in food sources, refuges and visibility. For water-birds, exposure to Phoslock® is likely to be restricted to the consumption of, for example, macrophytes, invertebrates and fish. However, as shown in studies with humans and other mammals, it is most likely that the amount of La adsorbed by waterbirds will be very low as most of the ingested La will be excreted with the faeces. For this reason, it seems unlikely that waterbirds will suffer any toxicity caused by Phoslock® and no adverse effects on the resident avian fauna has been observed during the numerous field applications. Pets such as cats and dogs could be exposed to Phoslock® treated water by swimming in or drinking treated water. Any risk from this exposure is evaluated from a range of data available for veterinary drugs and food additives using lanthanum carbonate.

Based upon the daily oral intake concentrations of La in pet food and drugs and the peak concentrations of TLa after Phoslock® treatment, even the consumption of lake water directly after the application would not pose a risk to animal health. iv

Humans may be exposed to Phoslock® during recreational activities via dermal contact and oral uptake by swallowing water or by consuming fish from treated water bodies. A huge amount of data is available from mammalian toxicity studies with Phoslock® and long term clinical studies with lanthanum carbonate, an FDA approved drug ingredient that is dosed up to 3 g La/day over many years. These data show a wide margin of safety associated with Phoslock® applications at predicted dosages and environmentally relevant concentrations. Ingestion of Phoslock® treated water even directly after an application would not pose any risk to human health. Conclusion In summary, testresults from acute and chronic toxicity studies support the conclusion that the use of Phoslock® is safe for the aquatic environment. The low ecotoxicological profile of Phoslock® is further confirmed through several field studies in which extensive post-treatment monitoring programs have been conducted following whole lake applications. Ecosystem changes are to be expected as Phoslock® will minimize cyanobaterial (blue-green algae) nuisance by reducing the P bioavailabilty. In doing so, however, the base of the food chain is altered towards a more desirable (less nutrient rich) ecosystem.

The use of Phoslock® as a tool for reducing eutrophication thus improves the trophic state of a waterbody, and thereby inducing a biological recovery process in formerly impaired aquatic systems. Expected changes in the Kralingse Plas For Kralingse Plas the calculated maximum dose of Phoslock® is within the range already applied in other systems. The application is recommended to be undertaken over several weeks, targeting different locations each day and the maximum total-La (TLa) concentration measured in the water column is expected to be similar to what has been measured in other aquatic systems (0.026 to 2.3 mg TLa/L). Based on the scientific literature discussed in this report and the Phoslock® dose calculated for Kralingse plas, a Phoslock® application to Kralingse Plas is not expected to result in any toxic effects in the aquatic organisms, nor any adverse effect on birds, pets and humans.

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Samenvatting Phoslock® In veel zoetwatermeren is eutrofiëring (vermesting) gaande. Om de beschikbaarheid van een overmaat aan fosfor (P) in het oppervlaktewater van dergelijke meren te verminderen en de waterkwaliteit te herstellen, zijn er zogenoemde geo-engineeringtools ontwikkeld. Eén van deze gereedschappen is het toedienen van het product Phoslock®, waarmee de nalevering van P naar de waterlaag kan worden verminderd. De ontwikkeling van Phoslock®, het juiste gebruik ervan en de dosering zijn allemaal gebaseerd op gedegen wetenschappelijke uitgangspunten. Phoslock® bevat 95% bentonietklei en 5% lanthaan. Lanthaan (La) is een van nature voorkomend element, dat in kleine hoeveelheden aanwezig is in sedimenten van Europese wateren. In Phoslock® is La sterk gebonden aan de bentonietklei, waarbij het tevens zijn capaciteit behoudt om fosfaat te binden. Zodra Phoslock® wordt toegepast in zoet water, verspreidt het zich als kleine deeltjes, bindt het P tot het onoplosbare lanthaan-fosfaat, waarna het snel bezinkt op de bodem. Lanthaan komt bij het toedienen van Phoslock® niet als oplosbaar La in het water vrij. De veiligheidsaspecten van Phoslock® hebben te maken met het actieve ingrediënt (La) en ook met de fysieke effecten. Fysieke effecten worden veroorzaakt door een tijdelijke toename van de troebelheid van het water tijdens de toediening.

In dit literatuuroverzicht is de evaluatie van het veilige gebruik van Phoslock® dan ook gebaseerd op de (ecotoxicologische) beoordeling van blootgestelde organismen in het aquatische ecosysteem en de blootstelling aan dieren op het land inclusief de mens. De effecten van La op aquatische organismen zijn getest in ecotoxicologische studies uitgevoerd met Phoslock® en oplosbare La-zouten (bv. LaCl3). Aangezien oplosbare La-zouten andere chemische eigenschappen hebben dan Phoslock®, zijn studies uitgevoerd met deze La-zouten niet vergelijkbaar met Phoslock® toepassingen. Tevens zijn deze studies vaak uitgevoerd onder niet-representatieve omstandigheden (zoals het gebruik van fosfaatvrij medium). Resultaten uit dergelijke onderzoeken zijn daarom niet geschikt om de toxiciteit van Phoslock® te beoordelen. Alleen ecotoxicologische testen uitgevoerd met Phoslock® vormen daarom de basis van dit literatuuroverzicht. Toxiciteit en biodiversiteitseffecten Tijdens een toepassing van Phoslock® zal de hoeveelheid zwevende deeltjes korte tijd toenemen. Deze tijdelijke toename kan een korte-termijn-impact hebben op organismen. Wanneer Phoslock® eenmaal is toegediend, zal het snel bezinken op de bodem van het meer, waarbij het merendeel binnen één dag bezinkt. Mogelijke ongewenste neveneffecten kunnen gemakkelijk voorkomen worden door de toediening te verspreiden over meerdere dagen.

Wanneer Phoslock® is toegepast zal het sterk en onomkeerbaar P binden, waardoor er minder P beschikbaar zal zijn voor de groei van (blauw)algen. Waargenomen negatieve effecten op de algengroei na toediening zullen dus eerder het gevolg zijn van de beperking van fosfaat dan van directe toxische effecten. Onderzoeken aan algen na toepassing van Phoslock® in hele meren tonen aan dat er als gevolg van de behandeling geen onbedoelde effecten op de algen zijn. De verwachte verschuiving na de behandeling van voedselrijke naar meer mesotrofe omstandigheden, met minder algenbiomassa, zal leiden tot een toename in algendiversiteit: minder cyanobacteriën (blauwalg) en een grotere diversiteit aan groene algen en diatomeeën. Dit gewenste effect wordt in het algemeen waargenomen na een sterke P-afname. Veranderingen in de voedselrijkdom van een meer zullen ook invloed hebben op andere gemeenschappen hoger in de voedselketen. Een van de gevolgen van minder fytoplankton is bijvoorbeeld een toename van het doorzicht in het water, wat een positief effect zal hebben op de waterplanten. Uit vele veldstudies blijkt inderdaad dat na een behandeling met Phoslock® het doorzicht toeneemt, gevolgd door een toename van de waterplanten (zie figuur hieronder).

Het bezinken van Phoslock® op bladeren zou sommige waterplanten enigszins negatief kunnen beïnvloeden, hoewel dit zeer onwaarschijnlijk is in een natuurlijk systeem, waar wind en golven de Phoslock® van de bladeren zullen afspoelen. Studies in het veld laten dan ook vi

zien dat er na de Phoslock® behandeling sprake is van een verhoogde bedekking met waterplanten, die gepaard gaat met een toename van het aantal soorten en de kolonisatiediepte. Toxiciteitsstudies met zoöplankton (zoals Daphnia) tonen aan dat met aanbevolen doseringen en onder realistische omstandigheden Phoslock® geen toxische effecten veroorzaakt. De resultaten laten ook een (zeer) ruime veiligheidsmarge zien voor in het veld toegepaste doseringen. Over het algemeen bleek in veldstudies waarin zoöplankton werd onderzocht, dat na de toepassing van Phoslock® de zoöplanktongemeenschap zich normaal ontwikkelde (zie figuur hieronder). Ook voor de in toxiciteitsstudies geteste macrofauna (zoals vlokreeften en muggenlarven) is er bij de toepassing van realistische Phoslock®-dosering sprake van een ruime veiligheidsmarge. Zelfs bij hoge testconcentraties werden geen significante effecten waargenomen bij de geteste soorten. Verschillende studies zijn uitgevoerd om de impact van Phoslock® op de ontwikkeling van benthische macrofauna in de gemeenschap te onderzoeken. Sommige studies vonden geen verschillen in macrofauna-gemeenschappen voor en na de behandeling. Andere studies vonden een (korte termijn-) effect op sommige groepen.

Veranderingen in de macrofaunagemeenschap kunnen worden toegeschreven aan de verandering van de voedselrijkdom van het meer, aangezien een toename aan waterplanten leidt tot een toename van de complexiteit van de habitat. Dit zal ook leiden tot een meer diverse macrofaunagemeenschap.

Zie Figuur 4, pagina 23: van links naar rechts (a) onbehandeld systeem met blauwalgenbloei in lente/zomer (b) systeem ondergaat Phoslock® behandeling in herfst/winter en (c) behandeld systeem in opvolgende lente/zomer.

De toxiciteitsstudies uitgevoerd met vissen laten eveneens een ruime veiligheidsmarge zien voor realistische Phoslock® doseringen. Op basis van deze studies worden geen risico's voor vissen verwacht bij toepassing van Phoslock® met standaard doseringen. De gerapporteerde en verwachte veranderingen in een toenemende bedekking aan waterplanten in het veld en de daaropvolgende macrofauna diversiteit kunnen een positieve invloed hebben op het visbestand. Er zal dan namelijk ook meer diversiteit zijn in voedselbronnen, schuilplaatsen en een beter doorzicht van het water. Voor watervogels is de blootstelling aan Phoslock® waarschijnlijk beperkt tot de consumptie van o.a. waterplanten, ongewervelden en vissen. Echter, zoals aangetoond in studies met mensen en andere zoogdieren, is het zeer waarschijnlijk dat de hoeveelheid geadsorbeerd La door watervogels zeer laag zal zijn, aangezien het meeste ingenomen La met de uitwerpselen zal worden uitgescheiden. Om deze reden lijkt het onwaarschijnlijk dat watervogels toxische effecten zullen ondervinden van Phoslock®. Ook zijn er geen nadelige effecten op de aanwezige vogelfauna waargenomen tijdens de vele veldtoepassingen. vii

Huisdieren zoals katten en honden kunnen in aanraking komen met water behandeld met Phoslock® door erin te zwemmen of ervan te drinken. De risico’s van deze blootstelling is geëvalueerd aan de hand van gegevens die beschikbaar zijn voor diergeneesmiddelen en voedseladditieven die gebruik maken van lanthaancarbonaat. Op basis van de dagelijkse orale inname van La in dierenvoeding en geneesmiddelen en de piekconcentraties van totaal-La (TLa) in water na Phoslock® behandeling, zou zelfs de consumptie van water direct na Phoslock® toediening geen risico vormen voor de gezondheid van de dieren. Mensen kunnen blootgesteld worden aan Phoslock® tijdens recreatie in het water via huidcontact en orale opname door water in te slikken, of door het eten van vis uit behandelde wateren. Er is een enorme hoeveelheid gegevens beschikbaar uit toxiciteitsstudies met zoogdieren en Phoslock®, en klinische studies uitgevoerd met lanthaancarbonaat, een medicijningrediënt, gedoseerd tot 3 g/dag elementair La over vele jaren. Deze gegevens laten een brede veiligheidsmarge zien ten aanzien van Phoslock® toepassingen bij normaal gebruik en dosering, en milieurelevante condities. Inname van met Phoslock® behandeld water, zelfs direct na toediening, zal geen risico vormen voor de menselijke gezondheid.

Conclusie Testresultaten uit acute en chronische toxiciteitsstudies ondersteunen de conclusie dat het gebruik van Phoslock® veilig is voor het aquatische milieu. Het lage ecotoxicologische profiel van Phoslock® wordt verder bevestigd door verschillende veldstudies waarbij een uitgebreid monitoringsprogramma werd uitgevoerd nadat het gehele watersysteem was behandeld. Omdat Phoslock® gericht is op het verminderen van de P-beschikbaarheid om blauwalgoverlast te minimaliseren, zijn veranderingen in het ecosysteem te verwachten. Hierdoor wordt de basis van de voedselketen veranderd naar een meer gewenst (minder nutriëntenrijk) ecosysteem. Het gebruik van Phoslock® als instrument voor het sterk verminderen van eutrofiëring verbetert dus de trofische toestand van een waterlichaam en brengt op deze manier een biologisch herstelproces op gang, van voorheen verstoorde aquatische systemen. Te verwachte veranderingen in de Kralingse plas Voor de Kralingse Plas ligt de berekende maximale Phoslock® dosis binnen de range van doseringen die reeds in andere systemen zijn toegepast. Het wordt aanbevolen om de toediening te verdelen over meerdere weken, gericht op verschillende locaties per dag, zodat de maximale gemeten TLa concentratie in het water naar verwachting vergelijkbaar zal zijn met wat is gemeten in andere aquatische systemen (0,026 tot 2,3 mg TLa/L).

Op basis van de wetenschappelijke literatuur, besproken in dit rapport, en de Phoslock® dosis berekend voor de Kralingse Plas, worden er geen toxische effecten verwacht op de aanwezige aquatische organismen als gevolg van een Phoslock® toediening, noch enig nadelig effect op vogels, huisdieren en mensen.

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Table of content 1.

Introduction ........................................................................................... 1 1.1. 1.2. 1.3.

2.

Toxicological assessment of Phoslock® .......................................................... 4 2.1. 2.2. 2.3.

3.

Phoslock® and its working mechanism ................................................................. 1 What is lanthanum? ........................................................................................ 2 Toxicology – Definition of terminology ................................................................. 2 Ecotoxicological assessment – Aquatic biota ........................................................... 4 Exposure scenario – Waterbirds and terrestrial mammals (particularly pets) ................... 16 Toxicological assessment – Human exposure to Phoslock® treated water ...................... 17

Reality check ........................................................................................ 19 3.1. 3.2. 3.3.

Kralingse Plas ............................................................................................. 19 Biological changes ....................................................................................... 20 Deferred eutrophication management ................................................................ 23

4.

Conclusion ........................................................................................... 25

5.

References ........................................................................................... 26

Appendix ................................................................................................... 32

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1. Introduction Phoslock® was developed in the nineties after rigorous scientific research undertaken by the Australia’s Commonwealth Scientific and Industrial Research Organisation (CSIRO) in order to remove phosphorus (P) from water bodies and restore water quality in aquatic systems suffering from eutrophication (Douglas, 2002). Until 2003 Phoslock® was produced as a pre-commercial slurry formulation. A granular prototype of Phoslock® was developed in 2004 (Robb et al., 2003; Martin & Hickey 2004) while the current commercial form of the product has been produced since the end of 2006. To assess and manage any possible adverse side effects on the environment and to humans of this phosphorus remediation technique, it is crucial to understand the hazards and risks associated with its use. This report aims to outline existing information on the product and assess any risks it may pose to the aquatic environment and humans. 1.1. Phoslock® and its working mechanism Phoslock® is a patented phosphorus locking technology containing 95% bentonite clay and 5% lanthanum (La) (Fig. 1). During the manufacturing process of Phoslock®, lanthanum cations are embedded inside the mineral layers of the bentonite clay, becoming the active ingredient.

Due to the charge imbalance on the surface of the clay mineral layers, the embedded lanthanum is strongly associated with the bentonite clay and, at the same time, retains its capacity to bind phosphate (Fig. 2). Lanthanum is not released as soluble lanthanum into the water when Phoslock® is applied in aquatic environments. The lanthanum will either react with phosphate or other oxyanions (e.g. carbonate) in the water body, or it will remain associated with the clay structure under a wide range of environmental conditions (Douglas et al., 2000; Dithmer et al. 2015, 2016; Reitzel et al., 2017). Lanthanum reacts preferentially with phosphate due to the strong affinity of lanthanum with phosphate.

Figure 1. On the left, a picture of Phoslock® as a dry material and on the right Phoslock® being mixed with lake water and applied on the surface of Lake de Kuil (the Netherlands).

Once in contact with phosphate, the La in Phoslock® will chemically inactivate P through precipitation (Fig. 2). Furthermore, it will form a mineral of low solubility called rhabdophane (LaPO4·n H2O, n ≤ 3) (Douglas et al., 2000; Slade and Gates, 1999), which over time will be transformed to monazite (LaPO4), a mineral with even lower solubility (Dithmer et al., 2015). Phoslock® can bind phosphate under almost all redox conditions (e.g. Meis et al., 2012; Mucci et al., 2018; Noyma et al., 2016; Waajen et al., 2016), in saline waters (Mucci et al., 2020), under a wide pH range (6 to 9) (Mucci et al., 2018; Li et al., 2019), and in the presence of high concentrations of humic substances (Dithmer et al., 2016a; Li et al., 2020). Once Phoslock® binds phosphate, the bound phosphate will not be released with redox changes, nor under natural variation in pH. Phosphate also remains bound during resuspension events (D’Haese et al., 2019; Douglas et al., 2016; Mucci et al., 2018). Theoretically, La- bound P will only be desorbed if pH drops below 4 or increases above 12 (Fig. 3A-Appendix). The P precipitated with Phoslock® will also not 1

be released in the presence of high amounts of DOC (Li et al., 2020). Therefore, phosphate bound to lanthanum in Phoslock® should be considered permanently unavailable for uptake by the aquatic organisms.

Bentonite layer

PO4 La

La

La

Figure 2. Scheme illustrating how the lanthanum embedded in the bentonite precipitated PO4 without being washed out from the clay (Mucci et al., 2020).

1.2. What is lanthanum? Lanthanum (La) is a naturally occurring element, classed in a group of elements commonly known as the rare earth elements (REEs). In the periodic table of elements, the REEs form a coherent chemical series from the atomic number 57 to 71 and include the elements yttrium (Y) and scandium (Sc). Despite the name, many REEs are more common than, for example, tin, copper and silver (Copetti et al., 2016). In the earth’s upper crust, lanthanum is present at a concentration of about 30 ppm and is most commonly found in minerals such as bastnäsite and monazite (BGS, 2011). In its pure form, lanthanum is a rather soft, silvery-white metal, which tarnishes rapidly in air. The chemistry of La is primarily based on the formation of the +3 oxidation state. La forms soluble salts with chloride, nitrate and sulphate anions, whereas lanthanum carbonate, -phosphate and -oxide are highly insoluble compounds. La is also found in aquatic systems where Phoslock® has not been applied; for instance background concentrations between 8 ng/L and 8.7 μg/L were reported from 18 groundwater and two surface water stations in the Netherlands and the reported background concentrations of lanthanum in European lake sediments range from 8 to 34 mg/kg dry weight (Cetiner et al., 2005; Meis et al., 2012; Nordberg et al., 2015; Yasseri & Epe, 2016). 1.3.

Toxicology – Definition of terminology Phoslock® is introduced into the environment as a geo-engineering tool, therefore it is essential to assess the potential risks associated with its use. Its development, appropriate use, and exact dose calculation are all based on solid scientific principles. Thus, any assessment of product safety should be described using the basic concepts of toxicological science. Before discussing the hazards and risks associated with the use of Phoslock®, the main terms commonly adopted in toxicology and used within this document will be introduced briefly: Hazard and Risk Hazard is defined as the inherent property of a substance to cause adverse effects under the conditions of exposure. In contrast, risk is the probability of occurrence of an adverse effect resulting from a given exposure to the substance.

2

Toxicological science Toxicology is the study of how chemicals interfere with the normal function of a biological system. Accordingly, the sciences concerned with the effects of substances on ecological systems or aquatic systems are referred to as ecotoxicology and aquatic toxicology, respectively (Sullivan et al., 2005; Calow & Forbes 2014). Dose descriptors The key principles in toxicology are exposure, dose, and dose-response relationships. In general, a toxicological assessment of adverse effects is based on defined dose descriptors, which are determined in specific acute and chronic toxicity studies with various sentinel organisms. During these tests, the exposure effects of a given substance on the test organisms, such as death, impaired swimming ability, failure of eggs to hatch or the ability to reproduce are noted and the values for the dose descriptors are statistically derived from the test data. Commonly the following dose descriptors are used:

•

LC50 dose (median lethal concentration, 50% of the test organisms die, endpoint death),

•

EC50 dose (median effective concentration, 50% of the test organisms show an effect, effect endpoints vary e.g., immobilization, reduction of growth parameters),

•

NOEC/NOAEL (no observed effect concentration; no observed adverse effect level)

•

LOEC/LOAEL (lowest observed effect concentration; lowest observed adverse effect level)

3

2. Toxicological assessment of Phoslock® The safe use of Phoslock® is related to its active ingredient lanthanum and, in the aquatic environment, also to physical effects caused by a temporary increase in turbidity in the water column (Spears et al. 2013a). Therefore the evaluation of the safe use of Phoslock® in this document is, based on: 1. The ecotoxicological assessment related to the specific features of the aquatic environment and biota to which it is introduced (chapter 2.1); 2. The evaluation of the exposure scenarios for terrestrial mammals and in particular pets (chapter 2.2); 3. The toxicological assessment related to human exposure to Phoslock® using the various services that aquatic ecosystems provide (chapter 2.3). 2.1. Ecotoxicological assessment – Aquatic biota Phoslock® has been applied to both lentic and lotic waterbodies. Both types of aquatic systems consist of two distinct zones: a pelagic zone consisting of the open water column and a benthic zone consisting of the sediments and the bottom waters. The following section provides an assessment of the environmental safety of Phoslock® based on a dose descriptor approach and its effect on suspended solids, bioaccumulation and biodiversity in pelagic and benthic habitats. 2.1.1.

Comparison of Dose Descriptors for a variety of aquatic biota- Laboratory studies Ecotoxicological laboratory studies on pelagic and benthic organisms have been conducted with both Phoslock® and soluble lanthanum salts (Copetti et al. 2016). These different stressors have produced a high variability in the determined dose descriptors due to the higher toxicological effects caused by the different chemical properties of soluble lanthanum salts compared to Phoslock®. Ecotoxicological assays for Phoslock® should reflect conditions which are representative of an application scenario and therefore tests conducted with Phoslock® itself and not La salts should form the basis of any ecotoxicity assessment. As a result, this approach has been adopted in this document. This approach is outlined in detail in box 1A of the appendix. Once Phoslock® is applied to freshwaters, it disperses into small particles. Lanthanum in the water column is measured analytically as either total lanthanum (TLa) or filterable lanthanum (FLa). Reitzel et al. (2017) found that Phoslock® disperses to the greatest degree in soft water lakes, producing fine clay particles with embedded La which pass through various filters with different pore sizes.

In the same study, the authors also measured FLa after 30 min ultracentrifugation, however fine colloids were still detected in the filtrate, demonstrating that the measured concentrations were not truly dissolved lanthanum. As a result, the authors concluded that FLa measured after ultracentrifugation could only be considered dissolved in low alkalinity water with very low phosphate and high dissolved organic carbon (DOC) concentrations. Yet, even here, the authors suggested that the measured FLa concentrations are probably associated with humic substances and not present as La3+ ions (Reitzel et al., 2017). Thus, TLa and FLa are an appropriate measure to estimate Phoslock® in the water column and assess its impact on the aquatic biota. The reported peak concentrations and the post-application range for TLa and FLa from 16 Phoslock® treated lakes are summarized in Table 1 (Spears et al. 2013a). The following section compares the dose descriptor values from tests for a range of aquatic biota with the reported peak TLa concentration in the water after applying Phoslock®. We have focussed on total lanthanum concentrations as filterable lanthanum concentrations are low after a Phoslock® application (for more detail about FLa in Kralingse Plas, see Paragraph 3.1).

The Netherlands is the only country in the world that has legislation relating to maximum permissible La concentrations (MPC) (Sneller et al., 2000) for surface water (10.1 µg FLa/L and 150 µg TLa/L) and for freshwater sediment (4700 mg TLa/ kg DW sediment). The MPC for La in surface water has been criticized in the scientific literature due to a number of methodological flaws (van Oosterhout et al., 2020). In addition, no experimental ecotoxicological tests with organisms were undertaken for the sediment data (Sneller et al., 2000). 4

Thus, in this report, we will discuss the effects of exposure to Phoslock® derived lanthanum based on a more recent set of ecotoxicological experiments conducted with different species in the water column and sediment (i.e., Table 2 to 7). Table 1. Reported peak concentrations and post-application range (up to 60 months) for total lanthanum (TLa) and filterable lanthanum (FLa) in different Phoslock® applications (Spears et al. 2013a).

Location

Timing

Peak TLa [mg/L]

Peak FLa [mg/L]

Water sampled from the lake surface

during and 1 month after application

0.026-2.3

0.002-0.414

Water sampled from bottom of the lake

during and 1 month after application

0.004-0.892

0.002-0.1

*

Surface-Bottom post application range (up to 60 months)

Range TLa [mg/L]

Range FLa [mg/L]

Surface

<0.001-0.031*

<0.001-0.002

Bottom

<0.001-0.068*

<0.001

0.031 and 0.068 mg/L were measured 6 months post-application.

Bacteria The EC50 values for the marine gram-negative bacterium Vibrio fischeri and nitrification rate in the sediment are 2173 and 15 times higher than the maximum reported concentration of TLa in lake water, respectively (Table 2). Thus, there are no toxic or direct impacts of Phoslock® on bacterial processes such as nitrification or the bacterial cell itself (Henriksen and Roslev, 2009 Garcia-Ordiales et al., 2019; Verweij et al., 2010). These results are also confirmed by a field survey undertaken in the Canning River after an application of Phoslock®. Sampling of both total heterotrophic bacteria and denitrifying bacteria showed no negative impact on the microbial communities in both the water and sediment, respectively (River Science 17, 2001). Table 2. Ecotoxicity studies with Phoslock® and bacterial activity or species. EC50 values (mg of Phoslock®/L) and the corresponding TLa concentrations.

Test organisms/process

Nitrification rate

Vibrio fischeri

Test conditions Phoslock® suspens. natural pond water Phoslock® suspens. natural pond water

Endpoint

Duration

EC50 Phoslock [mg/L]

Corresp. TLa [mg/L]

NO2 and NO3

48h

686

34.3

Light emission

30min

<25000

<5000

References

Henriksen and Roslev, 2009

Henriksen and Roslev, 2009

5

Phytoplankton It is important not to confuse indirect and direct effects when testing the toxicity of La to algae species. As La has a high affinity for phosphate, it binds quickly to any phosphate in solution, forming insoluble lanthanum phosphate, which precipitates in the culture medium. Thus, an observed adverse effect on algal growth could be due to phosphate limitation rather than direct toxic effects on the test species. This was demonstrated in a study by Petersen et al. (1974), who tested the effect of phosphate limitation in experiments conducted with the soluble lanthanum chloride (LaCl3) salt and two green algae species (Raphidocelis subcapitata, formerly Pseudokirchneriella subcapitata and Selenastrum capricornutum). No significant toxic effects up to a nominal concentration of 2 mg LaCl3/L were observed (as a soluble salt was used this corresponds to 1.1 mg ionic La). However, tests using soluble La compounds do not reflect the unique properties of Phoslock®, which is specially designed to not release soluble La forms in the water where it is applied. As results from toxicity testing of soluble La salts overestimate the adverse effects of Phoslock® derived La, these tests are inappropriate to assess Phoslock® effect on freshwater algae and therefore are not reported in this document.

In a laboratory test with a Phoslock® suspension under constant agitation, van Oosterhout & Lürling (2013) report reduced growth of two phytoplankton species (Scenedesmus obliquus; Microcystis aeroginosa) as well as effective control of a cyanobacteria bloom (Anabaena sp.). The control effect is attributed to the combination of phosphate binding, light limitation and flocculation of phytoplankton cells under conditions of prolonged exposure in the experiment rather than any toxic effect. In field conditions, the effect of light limitation and flocculation is expected to be less pronounced due to the rapid settling of the Phoslock® particles. This is also confirmed by a range of data from field applications analysing the algae assemblages after a Phoslock® treatment. Several studies found a shift in the phytoplankton community structure from cyanobacteria dominance to green algae and diatoms (Robb et al., 2003; Bishop et al., 2014; Bishop & Richardson 2018; Epe, 2014; Barçante et al., 2020). Phytoplankton surveys after whole lake applications of Phoslock® demonstrate that there are no non-target impacts on phytoplankton as a result of a treatment (Robb et al., 2003; Bishop et al., 2014; Bishop & Richardson 2018; Epe, 2014; Barçante et al., 2020).

Zooplankton Results from nine acute and chronic toxicity studies using two different water fleas species and a rotifer species are summarised in Table 3. The responses of these zooplankton species, including mortality, reproduction and growth, were evaluated throughout the Phoslock® exposures. The results show that for Daphnia magna, even when a concentration up to 10 times higher than the reported peak concentration of TLa ever found in a Phoslock® application is used, no adverse effect was observed (Lürling & Tolman 2010). In two other experiments using the same species (D. magna) similar results were observed, the NOEC was up to 500 (Martin and Hickey, 2004), and the EC50 between 100 (Watson and Leung, 2009) and >1087 (Martin and Hickey, 2004) times higher than the reported peak concentration of TLa ever found in a Phoslock® application (Spears et al. 2013a). Thus, the values show a wide margin of safety associated with Phoslock® applications at predicted use rates and environmentally relevant concentrations (cf. highest TLa reported during a Phoslock® application was 2.3 mg La /L, Spears et al. 2013a). Besides D. magna, the rotifer, Brachionus calyciflorus was evaluated in the presence of Phoslock®, showing that the population growth did not change even when double the maximum TLa concentration reported in a Phoslock® application was used.

Overall, this indicates that there is a wide safety margin when Phoslock® is applied under realistic conditions. During the early development phase of Phoslock® acute and chronic toxicity tests were conducted with Ceriodaphnia dubia using water samples from the Canning River after a Phoslock® treatment (Stauber & Binet, 2000). For this study two early formulations of Phoslock® were compared. It was demonstrated that modifications to the formulation to reduce the lanthanum release in surface water 6

resulted in the absence of toxicity. In this context it is important to point out that due to the amount of Phoslock® needed to impact biota, some studies did not use concentrations high enough to result in significant impacts. Thus, the respective toxicity endpoints in Table 3 are reported as being greater than the highest concentration tested and do not indicate the actual lowest observed effect levels. This was the case for the tests using the Ceriodaphnia dubia and therefore the EC50 values are not realistic and do not represent the actual Phoslock® concentration, which might influence the population. Table 3. Ecotoxicity studies with Phoslock® and zooplankton species. EC50 values and corresponding TLa concentrations. Table continues on next page.

Test organisms

Test conditions

Endpoint

Duration

NOEC EC50 Corresp. References Phoslock Phoslock TLa [mg/L] [mg/L] [mg/L]

Zooplankton Daphnia magna

Phoslock® Eluate, synth. soft water2)

Mortality

48 h

25000

> 500001)

1250; >2500

Martin & Hickey 2004

Daphnia magna

Phoslock® suspens. dechlor. tap water

Mortality

48 h

-

4900

245

WatsonLeung 2009

Daphnia magna

Phoslock® suspens. natural pond water

Mortality

48 h

-

>68003)

>340

WatsonLeung 2009

Phoslock® suspens. natural lake water

Mortality

48 h

50

> 501)

2.5; >2.5

ECOTOX 2008

River water after Phoslock® treatment

Immobility

48 h

n.s.

n.s.

>1.71)

Stauber& Binet 2000

154

5; 7.7

van Oosterhout & Lurling 2013

871-1557

5-25; 43.5577.85

Lürling Tolman 2010

Ceriodaphnia dubia

Ceriodaphnia dubia

Brachionus calyciflorus

Phoslock® suspens. WC medium

Popul. growth

Daphnia magna

Phoslock® suspens., artificial RT medium

Weight/ Length

48 h

5d

100

100-500

&

7

Test organisms

Ceriodaphnia dubia

Ceriodaphnia dubia

Ceriodaphnia dubia

Test conditions

Endpoint

Duration

NOEC EC50 Corresp. References Phoslock Phoslock TLa [mg/L] [mg/L] [mg/L]

Phoslock® suspens. natural lake water

Mortality

7d

1

> 11)

0.05; >0.05

ECOTOX 2008

Phoslock® suspens. natural lake water

Reproduction

7d

1

> 11)

0.05; >0.05

ECOTOX 2008

River water after Phoslock® treatment

Survival/Reproduction

7d

n.s.

n.s.

>1.71)

Stauber& Binet 2000

Due to the amount of Phoslock® needed to see impacts on biota, these studies did not test concentrations high enough to result in significant results; thus toxicity endpoints are reported as greater than the highest concentration tested and do not indicate the

1)

actual EC50 values. 2) Although tests using a Phoslock® suspension is the method of choice, results from eluate tests are included for comparison. 3 ) not high enough in the highest concentration, only 42 % of mortality.

Macrophytes No research has precisely calculated EC50 or NOEC values for Phoslock® on macrophytes. However, laboratory experiments have shown that Phoslock® does not reduce growth in Elodea nuttallii and Hydrilla verticillata (Chrzanowski, 2012; Wang et al., 2017) and does not hamper macrophyte germination when dosing up to 5 tons per ha (Waters-Hart, 2020) (Table 4). In another experiment, growth and responses of five other macrophyte species (i.e., Potamogeton perfoliatus, Myriophyllum spicatum, Elodea canadensis, Littorella uniflora , and Najas flexilis) were similar between the Phoslock® treatment and the control (Waters-Hart, 2020). This study also included an additional laboratory core experiment to test the impact of a Phoslock® treatment on submerged macrophyte leaves using the four macrophyte species Potamogeton perfoliatus, Myriophyllum spicatum, Elodea canadensis and Najas flexilis. The results show evidence of Phoslock® on the leaf surfaces (which was also confirmed through scanning electron microscope (SEM) imagery with X-ray analysis). However, the only species to respond negatively to the treatment in terms of its fluorescence (lower Fv/Fm values) was Potamogeton perfoliatus. Although this species showed a stressed state after exposure to Phoslock®, factors that can dislodge the product from the leaf surface in natural conditions (e.g.

wind, waves, fish and birds) were not present in the core experiment and need further investigation (Waters-Hart 2020). Nevertheless, the results from whole lake applications mentioned above show positive responses of the macrophyte community after a Phoslock® treatment and it can therefore be reasonably assumed that, under natural environmental conditions, negative effects on macrophytes will only be marginal as there is no indication for toxicity of Phoslock® to macrophytes.

8

Table 4. Ecotoxicity studies with Phoslock® and macrophyte species.

Test organisms

Test conditions

Duration

Dose used Phoslock [g/m2]

Corresp. TLa [g/m2]

Response

References

Macrophytes

Elodea nuttallii

Hydrilla verticillata

Phoslock® suspens. Synth. water

Phoslock® suspens. Filtered lake water

28d

30d

4968.1

Up to 20% of the sediment weight used

248.6

Plant biomass, shoot and root dry weight was not affected by Phoslock

Chrzanowski, 2012

unknown

In a low dose (2.5% of the sediment) the biomass decrease 20%. Higher dose up to 25 % of the sediment, the biomass was not affected.

Wang et al., 2017

Macroinvertebrates Results from toxicity studies using the freshwater shrimp Macrobrachium sp., the crayfish Procambarus fallax f. virginalis and sediment-dwelling invertebrates are given in Tables 5 and 6. The results indicated that the NOEC values for the decapod species were ten to thousand (Macrobrachium sp.) and two hundred (P. fallax f. virginalis) times higher than the reported peak concentration of TLa measured during a Phoslock® application (Spears et al., 2013a). Thus, there is a wide margin of safety (10 to 1087 times) associated with Phoslock® applications at predicted dose rates (c.f. Spears et al., 2013a). For the studies conducted with a range of sediment-dwelling organisms, different concentrations of Phoslock® suspensions were used. The concentration of Phoslock® near the benthic habitats is expected to be higher than in the overlying water. Nonetheless, the comparison of the corresponding TLa values derived from the Phoslock® exposure dose shows a wide safety margin, both in terms of the peak concentrations for TLa in lake bottom water (Table 1) and the dose descriptor values for the tested species (Table 6). At high test concentrations (>170 mg TLa/L), no significant survival or growth impacts were observed in any of the sediment toxicity test species (Table 6). Chironomus sp., for instance, was not affected in the presence of 10000 mg Phoslock/L (Fig.

4A-Appendix). This ensures that a considerable safety margin is maintained when Phoslock® is applied at the recommended dose rates. As was the case previously, the amount of Phoslock® needed to result in impacts to the test macroinvertebrates species was not high enough to result in significant impacts. Thus, the respective toxicity endpoints are reported as greater than the highest concentration tested and do not indicate the actual lowest observed effect levels.

9

Table 5. Ecotoxicity studies with Phoslock® and juvenile freshwater decapod species. EC50 values and corresponding TLa concentrations.

Test organisms

Test conditions

Endpoint

Duration

NOEC Phoslock [mg/L]

EC50 Phoslock [mg/L]

Corresp. TLa [mg/L]

References

Juvenile Decapods Freshwater shrimp: Macrobrachium sp. juvenile

Phoslock® Eluate, synth. soft water2)

Survival

96 h

50000

> 500001)

2500; >2500

Ecotox 2006

Macrobrachium sp. juvenile

Phoslock® Eluate, synth. soft water2)

Survival

7d

400

> 800

20; 40

Ecotox 2006

Macrobrachium sp. juvenile

Phoslock® Eluate, synth. soft water2)

Survival

14 d

400

800

20; 40

Ecotox 2006

Macrobrachium sp. juvenile

Phoslock® Eluate, synth. soft water2)

Survival

21 d

400

700

20; 35

Ecotox 2006

Macrobrachium sp. juvenile

Phoslock® Eluate, synth. soft water2)

Survival

28 d

400

700

20; 35

Ecotox 2006

500; >500

Lürling unpublished data; Fasola 2010

500; >500

Lürling unpublished data; Fasola 2010

Crayfish: Procambarus fallax f. virginalis

Phoslock® suspens. Copper free tap water

Procambarus fallax f. virginalis

Phoslock® suspens. Copper free tap water

Survival

Growth

96 h

28 d

10000

10000

> 100001)

> 100001)

Due to the amount of Phoslock® needed to see impacts to biota, these studies did not test concentrations high enough to result in significant results, thus toxicity endpoints are reported as greater than the highest concentration tested and do not indicate actual

1)

EC50 values. 2) Although tests using a Phoslock® suspension is the method of choice, results from eluate tests are included for comparison and due to lack of data from studies with Phoslock® suspension and shrimp.

10

Table 6. Ecotoxicity studies with Phoslock® and sentinel benthic macroinvertebrate species. EC50 values and corresponding TLa concentrations.

Test organisms

Test conditions

Endpoint

Duration

NOEC Phoslock [mg/L]

EC50 Phoslock [mg/L]

Corresp. TLa [mg/L]

Referen ces

Benthic Macroinvertebrates Amphipode: Hyalella azteca

Phoslock® suspens. natural pond water

Hyalella azteca

Phoslock® suspens. natural pond water

Growth

14 d

Hexagenia sp.

Phoslock® suspens. natural pond water

Survival

21 d

Hexagenia sp.

Phoslock® suspens. natural pond water

Growth

21 d

Chironomus dilutus

Phoslock® suspens. natural pond water

Survival

10 d

Chironomus dilutus

Phoslock® suspens. natural pond water

Chironomus sp.

Phoslock® suspens.

Survival

7d

Chironomus zealandicus

Phoslock® suspens. natural lake water

Survival

38 d

Survival

14 d

3400

3400

1)

170; > 170

WatsonLeung 2009

1)

> 3400

170; > 170

WatsonLeung 2009

> 4501)

22.5; > 22.5

WatsonLeung 2009

1)

> 450

22.5 > 22.5

WatsonLeung 2009

> 34001)

170; >170

WatsonLeung 2009

> 3400

170; >170

WatsonLeung 2009

>10000

500; >500

Lürling, unpublis hed data

> 4001)

20; > 20

Clearwat er 2004

> 3400

Mayfly: 450

450

Midge larvae:

Growth

10 d

3400

3400

10000

400

1)

Due to the amount of Phoslock® needed to see impacts to biota, these studies did not test concentrations high enough to result in significant results, thus toxicity endpoints are reported as greater than the highest concentration tested and do not indicate the

1)

actual EC50 values. 2) Although tests using a Phoslock® suspension is the method of choice, results from eluate tests are included for comparison

11

Fish Results from five acute toxicity studies using sentinel fish species are outlined in Table 7. A Phoslock® suspension of granules, Phoslock® treated river water and a Phoslock® eluate were used in these studies. As was the case with the zooplankton and macroinvertebrates species, a wide margin of safety was associated with Phoslock® applications at predicted use rates and environmentally relevant concentrations (cf. highest TLa reported during Phoslock® application was 2.3 mg La /L, Spears et al. 2013a). For instance, even when a concentration up to 60 times higher than the reported peak concentration of TLa ever found in a Phoslock® application is used, no adverse effect was observed in fish eggs as well as in the other fish species tested (Table 7). Based on these data, risks to fish are expected to be minimal with applications of Phoslock® at standard dose rates (cf. Spears et al. 2013a). Again, it is essential to mention that in two studies, the amount of Phoslock® used in the tests was not high enough to result in significant impacts on the fish, and an effective EC50 concentration could not be determined. Thus, the respective toxicity endpoints in Table 3 are reported as greater than the highest concentration tested. Table 7. Ecotoxicity studies with Phoslock® and sentinel fish species. EC50 values and corresponding TLa concentrations. n.s. = not specified.

Test organisms

Test conditions

Endpoint

Phoslock® granules, tap water

Coagulated eggs/heartbeat/anatomy /mortality

Duration

NOEC Phoslock [mg/L]

48h

-

EC50 Phoslock [mg/L]

Corresp. TLa [mg/L]

References

3000

150

Yasseri, 2008

Fish

Fish egg

Phoslock® Melanotaenia Eluate, 2500; Ecotox 2006 splendida > 500001) Mortality 96 h 50000 synth. soft >2500 water2) River water Melanotaenia Stauber& after Immobility 96h n.s. n.s. >1.71) duboulayi Binet 2000 Phoslock® treatment Phoslock® Oncorhynchus Martin & Eluate, 4350 156.3; mykiss Hickey 2004 Mortality 96 h <3125 synth. soft 217.5 2) water Phoslock® WatsonOncorhynchus suspens., > 136001) >680 mykiss Leung 2009 Mortality 48 h natural pond water 1) Due to the amount of Phoslock® needed to see impacts to biota, these studies did not test concentrations high enough to result in significant results, thus toxicity endpoints are reported as greater than the highest concentration tested and do not indicate actual EC50 values. 2) Although tests using a Phoslock® suspension is the method of choice, results from eluate tests are included for the purpose of comparison.

12

Summary In conclusion, by comparing NOEC and EC50 concentrations for various aquatic organisms with the reported TLa concentrations in lake water after an application of Phoslock®, it is apparent that even the highest TLa concentrations do not exceed the EC50 values of the various pelagic and benthic test organisms (Fig. 3). In the four studies conducted with the cladoceran species Ceriodaphnia dubia the respective Phoslock® concentrations used in the tests (1 mg/L and 50 mg/L, respectively) were too low to produce significant impacts to the test species. For this reason, an effective EC50 concentration could not be determined. The comparison of the reported values with other studies conducted with cladocerans indicates that higher effective EC50 concentrations can be expected, and, as a result, a sufficient safety margin could be assumed for Ceriodaphnia dubia. Furthermore, even though some of the studies do not report a NOEC for the Phoslock® exposure dose, the resulting EC50 concentrations are at least one order of magnitude (from 3 to 21739) higher than the measured peak TLa concentrations in lake water. Hence, based on the reported dose descriptor values for a representative range of aquatic organisms, the risk for aquatic life associated with the use of Phoslock® can be rated as low.

Vibrio Fischeri Nitrification rate Chironomus zealandicus Chironomus dilutus Chironomus dilutus Hexagenia sp Hexagenia sp Hyalella azteca Hyalella azteca Macrobrachium sp Macrobrachium sp Macrobrachium sp Macrobrachium sp Macrobrachium sp Procambarus fallax f. virginalis Procambarus fallax f. virginalis Fish egg Oncorhynchus mykiss Oncorhynchus mykiss Melanotaenia splendida Ceriodaphnia dubia Ceriodaphnia dubia Ceriodaphnia dubia Brachionus calyciflorus Daphnia magna Daphnia magna Daphnia magna Daphnia magna

>5000 >34 >20 >170 >170 >22.5 >22.5 >170 >170 35 35 40 40 >2500 >500 >500 150 217.5 680 >2500 > 0.5 > 0.5 > 2.5 >7.7 43 to 78 >340 245 >340

0

10

20

30

40

50

60

70

80

90

100

Total lanthanum concentration (mg/L)

Bacteria

May fly

Freshwater shrimp

Fish

Midge larvae

Amphipoda

Crayfish

Zooplankton

Figure 3. EC50 values indicated by numbers of lanthanum concentration (mg/L) for several aquatic organisms from different taxonomic groups (data from tables 2, 3, 4, 5, 6 and 7). The red vertical bar represents the maximum total lanthanum concentration measured during a Phoslock® application (2.3 mg T La/ L) (Spears et al., 2013a).

2.1.2. Suspended solids The estimated range of suspended solids during an application of Phoslock® is reported to be 0.62-46.0 mg/L. This temporary increase of suspended solids during the application of Phoslock® might exert a short-term impact, especially on pelagic organisms. However, once Phoslock® is applied, it will quickly 13

settle to the bottom of the lake. For instance, in Lake de Kuil (the Netherlands) 58% of the Phoslock® added settled within one day. After one week and four weeks, most of the Phoslock® had settled (88% and 94% respectively) (Mucci et al., in review). Similar TLa reduction over time was observed in lake Het Groene Eiland (The Netherlands) (Lürling & van Oosterhout 2013). Any potential effects from a temporary increase in suspended solids can easily be prevented in sensitive environments by adopting a targeted and site-specific approach, by controlling the quantity of Phoslock® applied daily, by tailoring the application strategy to each site and by avoiding applications during the growing season (Spears et al., 2013a). Application strategies should be defined once the final dose for each lake area is decided. 2.1.3. Bioaccumulation After applications of Phoslock®, lanthanum has been detected in macrophytes, chironomids, crayfish and fish, indicating the potential for bioaccumulation of Phoslock® derived lanthanum (van Oosterhout et al., 2020; van Oosterhout et al., 2014; Waajen et al., 2017b). However, no acute or chronic effects on the studied aquatic biota have been reported and no ecotoxicological effects have been observed for up to five years after Phoslock® applications.

Furthermore, lanthanum was mainly detected in the liver and hepatopancreas tissues of fish and aquatic invertebrates and the decreasing concentration patterns over time show depuration of lanthanum from the body of the aquatic biota (Landman et al., 2007; Waajen et al. 2017b). 2.1.4. Possible changes in biodiversity – Field observations Phoslock® is used to reduce excess phosphorus in water bodies suffering from anthropogenic eutrophication in order to mitigate against the negative effects caused by cyanobacterial blooms. As a result, several studies show that the shift in the trophic state of a waterbody towards more mesotrophic conditions leads to an increase in the biodiversity after an application of Phoslock® (e.g., Barçante et al., 2020; Bishop et al., 2014; Bishop and Richardson, 2018; Epe, 2014; Robb et al., 2003). Phytoplankton A Phoslock® application often leads to a shift in the phytoplankton community structure from bloomforming cyanobacteria dominance to much lower biomass of non-toxic green algae and diatoms. This reduced biomass and the increase of phytoplankton biodiversity is a desired post-treatment target and a commonly observed pattern after phosphorus reduction (Bishop et al. 2014; Schindler et al., 2016; Bishop & Richardson 2018). Lang et al.

(2016) have reported results from a field trial, documenting responses in phytoplankton community structure and abundance in Loch Flemington (UK). In this study, a Before-After-Control-Impact (BACI) analysis was used to distinguish natural variability from treatment effect and confirmed significant reductions in the magnitude of summer cyanobacterial blooms in Loch Flemington. Over the monitoring period, the phytoplankton community structure became less dominated by cyanobacteria and more functionally diverse. This resulted in continual improvements in the phytoplankton compositional and abundance metrics, which were not observed at the control site. However, this initial cyanobacterial response was not sustained beyond two years after application indicating that the applied quantity of Phoslock was insufficient to control internal P loading for longer than two years. The authors of this study emphasize that the effective control of internal P loading in Loch Flemington will require further implementation of both in-lake and catchment-based measures. Zooplankton Field studies in which zooplankton surveys have been undertaken show normal development of the zooplankton community after the application of Phoslock®.

However, in Lake Rauwbraken (The Netherlands) a temporary disappearance of the water flea Daphnia galeata was observed after the combined treatment of the flocculant poly-aluminiumchloride (PAC) and Phoslock® (Floc & Lock method). 14

Van Oosterhout & Lürling (2011) hypothesized that the reduction was likely caused by the physical effects of the aluminium-hydroxide flocs, grazing inhibition by flocs and suspended bentonite particles, low food concentrations and the absence of predation refuge due to high water transparency and not by toxic effects of La from Phoslock®. The Daphnia galeata population recovered three months after the Floc & Lock treatment (van Oosterhout & Lürling, 2011). In another Dutch lake (Lake de Kuil), the zooplankton community developed normally after Phoslock® treatment (Waajen et al., 2016a). Also, Meis (2012) did not find any changes in the abundance of individuals groups after a Phoslock® application in Loch Flemington (UK). Macrophytes In many eutrophic freshwaters, submerged macrophyte beds disappear due to the shift of the waterbody from a clearwater state with macrophyte dominance to a phytoplankton dominated turbid state (Scheffer & van Nes, 2007). Thus, mitigation of eutrophication should not only improve the chemical water quality but also promote the re-establishment of submerged macrophyte vegetation (Waajen et al., 2016a). Aquatic macrophyte community metrics after Phoslock® applications have been reported for several lakes in Europe, USA, and Australia (Bishop et al., 2014; Gunn et al., 2014; Novak & Chambers, 2014; Spears et al., 2016).