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The aquatic toxicity data submitted by the industry as full test reports are summarised in Table 3.3.1. The tests were conducted with freshwater organisms (bacteria, algae, crustaceans and fish).

Aquatic toxicity - microorganisms

In the two 16-h tests with the bacterium Pseudomonas fluorescens, in which two different grades of ZnO were tested (“Pharma A”, purity 99.9%, and “Rotsiegel”, purity 99.5%), no growth inhibition was observed up to the highest concentration tested, i.e. 100,000 mg ZnO/l, nominal concentration, equivalent to 80,000 mg Zn/l (Table 3.3.1: Institut Fresenius, 1989a,b). No reliable NOEC values can be derived from these tests because all test concentrations strongly exceeded the water solubility limit and actual dissolved zinc concentrations were not measured.

An activated sludge respiration inhibition test (OECD 209) was carried out with ZnO powder (LISEC, 1999b). The test was performed with activated sludge from a domestic STP and ZnO loading rates between 1 and 100 mg ZnO/l. The maximum tested loading rate of 100.4 mg ZnO/l, corresponding to 0.954 mg/l dissolved zinc, resulted in 7.9% inhibition. The EC50 for ZnO powder is therefore >100 mg ZnO/l, nominal concentration, equivalent to >80 mg Zn/l. In addition, an activated sludge respiration inhibition test (OECD 209) was done with tyre debris of cars (fraction < 100 μm) (LISEC, 1999c). The maximum tested loading rate of 99.4 mg/l tyre debris, corresponding to only 0.029 mg/l dissolved zinc, resulted in negligible inhibition (4.2%).

Aquatic toxicity - algae

The two tests with the unicelllular alga Pseudokierchneriella subcapitata (formerly known as Selenastrum capricornutum), in which two different grades of ZnO were tested (“Red seal-

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grade”, purity 99.77%, and “EPM-grade”, purity 99.37%), resulted in 72-h ErC50 values for

dissolved zinc of 135 and 136 µg Zn/l, respectively, for endpoint specific growth rate. The 72-h NOErC values for dissolved zinc were 8 and 24 µg/l, respectively (Table 3.3.1: LISEC,

1997; Van Ginneken, 1994a). These NOEC values suggest that Red seal-grade ZnO may be somewhat more toxic than EPM-grade ZnO, but because of some differences between the two tests (using either statistics to derive the NOEC or using the lowest test concentration that resulted in less than 10% effect as NOEC; and either measuring dissolved zinc in the stock solution or in the test waters) and the small difference between the NOEC values, a firm conclusion cannot be drawn. Although red-seal grade ZnO and EPM-grade ZnO both have a high purity, the former contains somewhat less impurities (soluble salts) and is somewhat less soluble than the latter (see also footnote 7 below Table 3.3.1). Based on these characteristics, a somewhat lower toxicity could be predicted for Red-seal ZnO compared to EPM-grade ZnO, which seems to be not in agreement with the above test results.

It is noted that similar growth inhibition tests with the same algal species have been conducted with either a soluble zinc compound or with zinc metal powder (see Table 3.3.2.a and Table 3.3.2.d, respectively, in Annex 3.3.2.A of the Risk Assessment Report on Zn metal). These tests and the above tests with ZnO, all using soft to very soft artificial test media, resulted in comparable NOEC values if expressed as dissolved zinc, i.e. NOEC values in the range of 5-50 µg/l, regardless whether a soluble or “insoluble” test compound was used.

Aquatic toxicity - invertebrates

A short-term Daphnia magna immobilisation test with “EPM-grade” ZnO (purity 99.37%) resulted in a 48-h EC50 for dissolved zinc of 1,760 µg/l and a 48-h NOEC for dissolved zinc of 280 µg/l (Table 3.3.1: Van Ginneken, 1994b).

It is noted that the 48-h NOEC of 280 µg/l from this short-term test is within a factor of 2 of a number of NOEC values (endpoints: survival, reproduction and/or growth) derived in long- term D. magna tests in which a soluble zinc salt was used as test compound (see Table 3.3.2.a in Annex 3.3.2.A of the Risk Assessment Report on Zinc metal).

Aquatic toxicity - fish

In a 96-h acute toxicity test with fish Brachydanio rerio (test compound “EPM-grade” ZnO, purity 99.37%), no effect was found for dispersed ZnO at 100 mg ZnO/l (limit test), thus the 96-h EC50 is >100 mg ZnO/l, nominal concentration, equivalent to >80 mg Zn/l. The actual dissolved zinc concentration in this ZnO dispersion was 4,700 µg Zn/l (Table 3.3.1: Van Woensel, 1994b).

Aquatic toxicity - amphibians

For tadpoles of the amphibian Bufo bufo japonicus exposed to ZnO, a 48-h EC50 for dissolved zinc of 3,200 µg Zn/l has been reported (static test, at pH 7.6); the toxicological endpoint was not reported (ZnO IUCLID data sheet, ECB-version of 28 March 1995).

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Table 3.3.1 Toxicity of zinc oxide to freshwater organisms: NOEC and EC50 values

--- Organism & Test compound Test- pH Hard- Exp.- Criterion Result Result

life stage & purity water ness time (mg ZnO/l) (mg Zn/l) --- Bacteria

Pseudomonas fluorescens ZnO (Pharma A) art. 7.0 - 16-h NOECg >100,000 >80,000 (Cn)

99.9% Institut Fresenius ‘89a [1]

Pseudomonas fluorescens ZnO (Rotsiegel) art. 7.0 - 16-h NOECg >100,000 >80,000 (Cn)

99.5% Institut Fresenius ‘89 b [1]

Algae (unicellular)

Selenastrum capricornutum ZnO (Red seal-grade) art. 8.5 12 72-h ErC50g 0.17 0.135 (Cn-d)

99.77% EbC50g 0.043 0.034 (Cn-d)

NOErCg 0.010 0.008 (Cn-d)

NOEbCg < 0.005 < 0.004 (Cn-d)

LISEC ‘97 [2,7]

Selenastrum capricornutum ZnO (EPM-grade) art. 7.5 24 72-h ErC50g 0.17 0.136 (Cd)

99.37% NOErCg 0.03 0.024 (Cd)

Van Ginneken ‘94a [3,6,7] Crustaceans

Daphnia magna ZnO (EPM-grade) art. 7.7 261 48-h EC50i 2.2 1.76 (Cd)

age <24 h 99.37% NOECi 0.35 0.28 (Cd)

Van Ginneken ‘94b [4,6,7]

Fish

Brachydanio rerio ZnO (EPM-grade) art. 7.9 266 96-h NOLC >5.9 >4.7 (Cd)

length 3.64 ± 0.21 cm 99.37% Van Woensel ‘94b [5,6,7]

--- All tests: static test system, except the test with fish Brachydanio rerio (conducted in a circulation system).

g = growth (r: growth rate; b: biomass) Cn: Nominal concentration in test water.

Cn-d: Nominal dissolved concentration in test water, based on analyses of zinc in the 0.1 µm filtered stock solution Cd: Measured dissolved concentration in test water, based on analyses of zinc in 0.45 µm filtered test waters.

[1] No statistics reported. Two lots of ZnO were tested, produced by PHARMA and ROTSIEGEL, respectively. Tests carried out according to Bringmann (1973). In both tests, no growth inhibition was observed up to the highest test concentration (100,000 mg ZnO/l, equivalent to 80,000 mg Zn/l) and all test concentrations (100 - 100,000 mg ZnO/l) strongly exceeded the maximum water solubility of 1.6 mg/l reported for ZnO by Weast (1974). See also below for data on the water solubility of ZnO.

Dissolution procedure for preparing the stock solution: no data. Particle size <42 µm.

[2] Statistics: applied to derive EC50 and NOEC values. Test compound: Red Seal-grade ZnO; diameter (d50): 0.57 µm. Test conducted according to OECD-guideline 201 and under GLP. Algal medium according to OECD-guideline No. 201 (nominal background zinc concentration: 1.5 µg/l; hardness 24 mg/l (as CaCO3)), but EDTA was omitted. Nominal test concentrations: 0-3.7-8-18-40-87-192 µg

dissolved-Zn/l, using a dilution factor of 2.2. The dilutions were prepared as follows: a filtrate (0.1 µm filter) of a 100 mg ZnO/l dispersion in demineralized water was diluted with demineralized water up to 2-times the required test concentration and further 1:1 diluted with the algal suspension, thus reducing the background zinc concentration and hardness of the test medium to about 0.8 µg/l and 12 mg/l, respectively. Reported nominal dissolved-zinc concentrations in test water: based on analyses of zinc in the 0.1 µm filtered stock solution. The algal preculture, used for the inoculation of the test medium, was incubated under the conditions of the test for 3 days.

Toxicological endpoint: growth (biomass) and specific growth rate (measured by cell density). At the LOEC for biomass (dissolved-Zn concentration 0.018 mg/l, equivalent to 0.023 mg ZnO/l), 30% inhibition of this endpoint was observed. At the LOEC for growth rate (dissolved-Zn concentration 0.040 mg/l, equivalent to 0.050 mg ZnO/l), 18% inhibition of this endpoint was observed.

Results reported as nominal dissolved-Zn concentration, calculated from the dissolved-Zn conentration measured in the 0.1 µm filtered stock solution. The nominal test concentrations have been confirmed by actual zinc analyses in the test waters. At nominal dissolved-Zn concentrations of 0 (control), 0.004, 0.008 and 0.018 mg Zn/l, the measured dissolved-Zn concentrations were below 0.008 mg/l (detection limit), both at start and end of the test, regardless of the presence of algae in the water (except for the 0.0018 mg/l concentration which decreased from 0.013 mg/l at start to < 0.008 mg/l at end). In the highest three concentrations, the dissolved-Zn concentrations measured at the end of the test were lower in the test waters with algae than in the test waters without algae (reference test waters) due to uptake/adsorption of zinc by algae. The dissolved-Zn concentrations in the reference test waters remained, however, within 80% of the initial concentration throughout the duration of the test.

Dissolution procedure for preparing the stock solution (100 mg ZnO/l dispersion): stirring on a magnetic stirrer for 3 days at room temperature.

If using NOEC = the lowest test concentration that resulted in less than 10% effect (as applied in the test with Selenastrum capricornutum by Van Ginneken, 1994a), the NOEC for growth rate would be 0.018 mg Zn/l and the NOEC for biomass would be 0.008 mg Zn/l.

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[3] No statistics reported. Test conducted according to OECD-guideline 201 and under GLP. Test medium according to OECD-guideline No. 201, but EDTA was omitted. Test medium sterile-filtered (0.45 µm filter) before use in test. In the test, a control, a filtrate (0.45 µm filter) of a 100 mg ZnO/l dispersion and a series of four dilutions of the filtrate were tested, using a dilution factor of 3.2. If the concentration of the test substance in the filtrate is expressed as 100%, then the following dilutions were tested: 31.25%, 9.76%, 3.05% and 0.95%. Toxicological endpoint: specific growth rate (measured by cell density). Based on the aforementioned “nominal” concentrations, the 72-h EC50, 72-h LOEC and 72-h NOEC were 19.69%, 9.76% and 3.05% of the concentration in the filtrate, respectively. At the LOEC (actual concentration 0.08 mg Zn/l, equivalent to 0.1 mg ZnO/l), 22% inhibition of the specific growth rate was observed.

Actual dissolved background zinc concentration in test medium after 72 hours: 0.024 mg Zn/l (equivalent to 0.03 mg ZnO/l). It is noted that after 72 hours, the 0.95% en 3.05% dissolution of the filtrate (the latter value being the NOEC) contained the same actual dissolved zinc concentration as the control medium. Also the actual dissolved concentrations averaged over the 72-h exposure period (average of 0-h and 72-h measurement) were practically the same in these three groups, varying from 0.016 to 0.024 mg Zn/l (0.02 to 0.03 mg ZnO/l). Actual dissolved concentrations: based on measurements of dissolved zinc (0.45 µm filter); the values listed in Table 3.2.1 are based on measurements after 72 hours.

Dissolution procedure for preparing the stock solution (100 mg ZnO/l dispersion): no data.

[4] Statistics: only applied for calculation of EC50. Test conducted according to OECD-guideline 202 and under GLP. Test medium according to EEC standard No. L.251/146 Part C2, 1.6.1.2. annex (1984), to which micro-nutrients were added, but EDTA was omitted. In the test, a control, a filtrate (0.45 µm filter) of a 100 mg ZnO/l dispersion and a series of four dilutions of the filtrate were tested, using a dilution factor of 3.2. If the concentration of the test substance in the filtrate is expressed as 100%, then the following dilutions were tested: 31.25%, 9.76%, 3.05% and 0.95%. Toxicological endpoint: immobilisation. Based on the aforementioned “nominal” concentrations, the 48- h EC50, 48-h LOEC and 48-hr NOEC were 65.2%, 100% and 9.76% of the concentration in the filtrate, respectively. At the LOEC (actual dissolved concentration 2.7 mg Zn/l, equivalent to 3.4 mg ZnO/l), 17 out of 22 dapnids were immobile.

Actual dissolved background zinc concentration in test medium: 0.045 mg/l (equivalent to 0.056 mg ZnO/l). Actual dissolved concentrations: based on measurents of dissolved zinc (0.45 µm filter); averages of 0-h and 48-h measurement.

Dissolution procedure for preparing the stock solution (100 mg Zn/l dispersion): stirring on a magnetic stirrer for 24 hours.

[5] No statistics reported. Limit test conducted according to OECD-guideline 203 and under GLP. Test medium according to EEC-guideline 79-831, Annex V, part C.1. (1984). In the test, a control, a 100 mg ZnO/l dispersion and a filtrate (0.45 µm filter) of a 100 mg ZnO/l dispersion were tested. No effects on survival and behaviour were observed in any group.

Actual dissolved background zinc concentration in test medium: 0.024 mg/l (equivalent to 0.03 mg ZnO/l). Actual dissolved concentrations in filtrate and dispersion: 1.06 mg Zn/l (1.33 mg ZnO/l) and 4.7 mg Zn/l (5.9 mg ZnO/l), respectively. Actual dissolved concentrations: based on measurements of dissolved zinc (0.45 µm filter); averages of 0-h and 96-h measurement. The measured total concentration in the dispersion was 17.9, 11.3 and 9.0 mg Zn/l (22.4, 14.1 and 11.2 mg ZnO/l) at the 0-h, 24-h and 96-h measurement, respectively, indicating that it was impossible to take a representative sample of the dispersion.

Dissolution procedure for preparing the stock solution (100 mg ZnO/l dispersion): no data.

[6] Test compound: EPM-grade ZnO (“direct oxide”)(batch 193031). Purity 99.37%; Impurities include 0.25% water soluble zinc salts which are dissolved over time, in addition to a rapid dissolution of pure ZnO which takes place up to the concentration of the solubility product of ZnO (Jahn, 1997).

[7] According to Jahn (1997), the EPM-grade ZnO is not representative of the most common type of zinc oxide produced: more than 70% of the total ZOPA (Zinc Oxide Producers Association) production is Red Seal-grade ZnO (“indirect oxide”). Red seal-grade ZnO contains virtually no soluble salts.

Jahn (1997) includes an abstract of the draft report “Transformation/dissolution of zinc oxide powders in ecotox media”, with the results of a 4-d dissolution study with Red Seal-grade ZnO and a 16-d dissolution study with EPM-grade ZnO, both in “modified algal medium” (background dissolved zinc concentration up to 0.008 mg/l):

The data for Red Seal-grade ZnO show that nominal concentrations of 1 to 500 mg ZnO/l “modified algal medium” resulted in dissolved (0.2 µm filter) zinc concentrations of 0.3 to 0.4 mg Zn/l in 4 days. The 4-d dissolution curves for Red Seal-grade ZnO show an initial rapid increase in dissolved zinc concentrations (especially in the first hours) and almost equilibrium in 4 days, regardless of the nominal concentration.

The data for EPM-grade ZnO show that nominal concentrations of 1 to 500 mg ZnO/l “modified algal medium” resulted in dissolved zinc concentrations of 0.4 to 0.9 mg Zn/l in 4 days and dissolved zinc concentrations of 0.7 to 1.8 mg Zn/l in 16 days. The 16-d dissolution curves for EPM-grade ZnO also show a rapid initial increase in dissolved zinc concentrations, but at the higher concentrations (100 and 500 mg ZnO/l) a slow but steady further increase after day 4.

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3.3.1.1.2 Terrestrial compartment