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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the target substance Ferbam and the source substance Ziram quickly hydrolyse forming dimethyldithiocabamates. The analogue hypothesis is that both substances dissociate in acidic and neutral aqueous solutions to the common breakdown product dimethyldithiocarbamate, which determines the (eco-)toxicological properties. Therefore, the basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach (Read-Across Assessment Framework, RAAF, ECHA, 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance is Ziram (IUPAC name: zinc bis(dimethyldithiocarbamate , CAS no. 137-30-4; EC no. 205-288-3). Ziram is an organometallic mono-constituent substance with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The target substance is Ferbam (IUPAC name: N,N-dimethylcarbamodithioate;iron(3+), CAS no. 14484-64-1; EC no. 238-484-2). Also Ferbam is an organometallic mono-constituent substance, with a typical purity of ca. 99% (w/w), ranging from > 98% to 100%. In general, impurities are known and are likely to vary depending on the manufacturing process, but are considered not relevant for classification or labelling of the substance.
The substance identities and all REACH Annex VII and VIII information on physicochemical properties, environmental fate, ecotoxicity and toxicity are summarised in the read-across statement attached under section 13 of this IUCLID file. Both substances are dimethyldithiocarbamates with a central metal. The high structural similarity is also reflected in very similar physicochemical properties. Both substances are primarily used fungicides.

3. ANALOGUE APPROACH JUSTIFICATION
The toxicity of Ziram to algae has been investigated in a growth inhibition test according to OECD TG 201. In the key study (Dobbins, 2014) measured concentrations ranging from 12 to 180 µg/L were tested. After 72 h of exposure, the EC50 based on growth rate was determined as 94 µg/L. Due to the similar hydrolysis, the read-across of the studies and their results is justified, as substances are applied in dilution to water.
For further information on the justification of the analogue approach please refer to the read-across statement attached under section 13 of this IUCLID file.

4. DATA MATRIX
Please refer to the data matrix in the read-across statement attached under section 13 of this IUCLID file.
Reason / purpose:
read-across source
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test conditions:
Test Apparatus
Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers, and contained 100 mL of test or control medium. The test chambers were labeled with the project number, test concentration and replicate, and were indiscriminately positioned daily in an environmental chamber designed to maintain the desired test temperature throughout the test. The test flasks were continuously shaken on a mechanical shaker at 100 rpm throughout the duration of the test.

Environmental Conditions
Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion Model 4Star plus pH/ISE meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the remaining replicates of each treatment and control group.

Preparation of Test Concentrations
A stock solution was prepared by dissolving 0.0304 g (corrected for the given purity of 98.7%) of the test substance in 10 mL of N,N-dimethylformamide (DMF; Burdick and Jackson Brand, >99.99% pure) to achieve a nominal concentration of 3000 μg a.s./mL. The 3000 μg a.s./mL stock was mixed by inversion and appeared clear and slightly tan in color after mixing. The 3000 μg a.s./mL stock was serially diluted with DMF to prepare four additional stocks at target nominal concentrations of 188, 375, 750 and 1500 μg a.s./mL. Each test solution was prepared by diluting 100 μL of each respective stock in 1000 mL of freshwater AAP medium. Test solutions were prepared using this method to ensure that the solvent concentration in each treatment group was equivalent. All of the test solutions appeared clear and colorless after mixing. The negative control solution consisted of freshwater AAP medium without test substance added. The solvent control solution contained 0.1 mL DMF/L, which was equivalent to the solvent concentration in all of the treatment groups.

Inoculation of Test Chambers
Prior to test initiation, the concentration of algal cells in the stock culture (culture identification No. 00-1) was determined using a hemacytometer and a microscope, and was 4.10 x 106 cells/mL. In order to achieve the desired initial cell density of 10,000 cells/mL, 0.244 mL of stock culture was added to each replicate test chamber at test initiation using an Eppendorf pipette.

Algal Growth Measurements and Observations
Test medium samples were collected from each replicate of the treatment and control groups for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure and were held for a maximum of one day under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Pseudokirchneriella subcapitata. A primary counting standard containing Pseudokirchneriella subcapitata cells was prepared at a target nominal concentration of 100,000 cells/mL. The density of the primary counting standard was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e., the R-squared values obtained through the regression analysis were ≥ 0.99922). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton®). Three 0.5-mL volumes of the diluted sample were counted, and the resulting counts were averaged. The cell density of the sample was determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, based upon the Y-intercept and slope calculated through the regression analysis, and the dilution factor. The following equation was used:
Cell Density of Sample (cells/mL) = (((Mean Cell Count (cells/mL)) - (Y Intercept)) / Slope) * Dilution Factor
Samples of test solution were collected from each replicate at the end of the test. These samples were pooled within their respective treatments, and sub-samples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregation or flocculation of cells, and adherence of the cells to the test chamber.

The calculation of cell densities, yield, growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows, Version 8.2” (7). Growth rate was calculated for each replicate of the control and treatment groups at 24, 48 and 72 hours using the following formula:
μ = (ln Nn - ln No) / (tn -to)
where: μ = Average specific growth rate
No = Nominal cell density (cells/mL) at to
Nn = Measured cell density (cells/mL) at tn
to = Time of beginning of test (hours)
tn = Time after beginning of test (hours)
Yield was calculated for each replicate of the control and treatment groups at 72 hours as the final cell density in the exposure period minus the initial nominal cell density. Inhibition values were calculated for each treatment group as the percent reduction in yield and growth rate relative to the negative control replicates using the following formula:
Percent Inhibition = ((Mean Control Response - Mean Treatment Response) / Mean Control Response) x 100
ECx values in this study represent the theoretical test concentrations that would produce an x% reduction in a variable of interest relative to the control. The ECx values and their corresponding 95% confidence intervals were calculated, when possible, using non-linear regression (8) with treatment response (yield and growth rate) and geometric mean, measured test concentrations. The negative and solvent control responses for yield and growth rate were compared using a t-test (α = 0.05). No significant differences were detected, therefore the controls were pooled for comparison to treatment responses. The 72-hour yield and growth rate data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. Mean treatment group responses were compared to the pooled control response using Dunnett’s one-tailed t-test (α = 0.05). The results of the statistical analyses were used to determine the NOEC relative to each parameter at 72 hours.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
73.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
94 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
53.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
53.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Details on results:
Measurement of Test Concentrations
Nominal test concentrations were 18.8, 37.5, 75, 150 and 300 μg a.s./L. The 150 and 300 μg a.s./L samples were diluted 5-fold to bring the concentrations within the calibration range. The test solutions appeared clear and colorless at the time of preparation, and there was no evidence of surface slicks or precipitation at exposure termination. Measured concentrations of Ziram on day 0 ranged from 86 to 114% of nominal. Measured concentrations of Ziram in the test solutions on day 1 ranged from 59 to 81% of nominal, and on day 2 ranged from 47 to 58% of nominal. Measured concentrations of Ziram in the test solutions at test termination ranged from 41 to 67% of nominal. The results of the study are based on geometric mean, measured concentrations of 12.0, 27.8, 53.4, 97.4 and 180 μg a.s./L, representing 64, 74, 71, 65 and 60% of the nominal test concentrations.

Observations and Measurements
Temperatures remained within the 24 ± 2°C range established for the test. The pH of the test solutions ranged from 7.3 to 7.4 at test initiation and ranged from 7.4 to 9.0 in the test solutions at test termination. The observed increase in pH is typical for tests conducted with P. subcapitata and is attributed to the photosynthetic activity of the algae. The light intensity ranged from 5,540 to 6,120 lux, which was within the desired range of 6000 lux ± 10%.
The toxicity of Ziram to P. subcapitata was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to calculate growth rates for each 24-hour interval of exposure and yields at 72 hours of exposure. Exponential growth, characterized by the linear section of the growth curve, occurred throughout the duration of the test in the negative control replicates.
After 72 hours of exposure, inhibition of yield in the 12.0, 27.8, 53.4, 97.4 and 180 μg a.s./L treatment groups was 16, -1, 4, 95 and 100%, respectively, relative to the pooled control. Inhibition of growth rate in the 12.0, 27.8, 53.4, 97.4 and 180 μg a.s./L treatment groups was 4, 0, 0, 56 and 99%, respectively, relative to the pooled control. Dunnett’s test indicated mean yield and growth rate were significantly reduced (p <0.05) in the 97.4 and 180 μg a.s./L treatment groups when compared to the pooled control. Therefore, the 72-hour NOEC for yield and growth rate were both determined to be 53.4 μg a.s./L. The 72-hour EC50 values for yield and growth rate were determined to be 73.3 μg a.s./L and 94.0 μg a.s./L, respectively.
After 72 hours of exposure, adherence of cells to the test chambers or aggregations of cells were not observed in any of the control or treatment groups. Cells in the solvent control and treatment groups appeared normal when compared microscopically to cells present in the negative control.

Conditions for the Validity of the Test
Mean cell density in the control flasks increased by a factor of 206 after three days, achieving the 16x growth criterion. The coefficient of variation of average specific growth rates in the control replicates was 2.7%, achieving the less than 7% criterion. The mean percent coefficient of variation for section-by-section specific growth rates in the control replicates was 21.3%, achieving the less than 35% criterion.
Validity criteria fulfilled:
yes
Conclusions:
The freshwater alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five treatment levels of Ziram ranging from 12.0 to 180 mg a.s./L, based on geometric mean, measured concentrations of Ziram. Toxicity of Ziram to P. subcapitata was assessed based on effects on growth rate and yield relative to the pooled control. The 72-hour EC50 values for yield and growth rate were determined to be 73.3 μg a.s./L and 94.0 μg a.s./L, respectively. The 72 hour NOEC values for growth rate and yield were both determined to be 53.4 μg a.s./L.
Executive summary:

STUDY: Ziram: A 72-Hour Toxicity Test with the Freshwater Alga (Pseudokirchneriella subcapitata)

TEST GUIDELINES: OECD Guidelines for Testing Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test; and the Official Journal of the European Communities No. L383 A, Method C.3: Algal Inhibition Test; GLP

LENGTH OF EXPOSURE: 72 Hours

TEST ORGANISM: Freshwater Alga (Pseudokirchneriella subcapitata)

The freshwater alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five treatment levels of Ziram ranging from 12.0 to 180 mg a.s./L, based on geometric mean, measured concentrations of Ziram. Toxicity of Ziram to P. subcapitata was assessed based on effects on growth rate and yield relative to the pooled control.

RESULTS:

Endpoint 0-72 Hours*
Yield:
EyC10 58.8 μg a.s./L
95 % Confidence Interval 12.5 to > 180 μg a.s./L
EyC20 63.4 μg a.s./L
95 % Confidence Interval 16.9 to > 180 μg a.s./L
EyC50 73.3 μg a.s./L
95 % Confidence Interval 30.2 to 178 μg a.s./L
NOEC 53.4 μg a.s./L
Growth Rate:
ErC10 65.8 μg a.s./L
95 % Conficence Interval 60.9 to 71.2 μg a.s./L
ErC20 74.4 μg a.s./L
95 % Confidence Interval 69.8 to 79.2 μg a.s./L
ErC50 94.0 μg a.s./L
95 % Confidence Interval 90.0 to 97.7 μg a.s./L
NOEC 53.4 μg a.s./L
* Base onf geometric mean measured test concentrations of Ziram. Ecx values were calculated using non-linear regression. NOEC values were based on statistical comparisons (Dunnett's test; p < 0.05) between treatment and pooled control data.

Description of key information

Read-across

ErC50 (72h) = 94 µg/L for Pseudokirchneriella subcapitata

NOEC (72h) = 53.4 µg/L for Pseudokirchneriella subcapitata

Key value for chemical safety assessment

EC50 for freshwater algae:
94 µg/L
EC10 or NOEC for freshwater algae:
53.4 µg/L

Additional information

In the key study toxicity of read-across source substance Ziram to Pseudokirchneriella subcapitata was assessed based on effects on growth rate and yield relative to the pooled control. The 72-hour EC50 values for yield and growth rate were determined to be 73.3 μg a.s./L and 94.0 μg a.s./L, respectively. The 72 hour NOEC values for growth rate and yield were both determined to be 53.4 μg a.s./L.

In a supporting study with Ferbam the derived 96-hour EC50 (based on the growth rate) was determined to be 2.4 mg/L nominal for Chorella pyrenoidosa. The lowest derived result of the parametric analysis is 0.33 mg/L for the effects of the inoculum.