Zinc bis(dibenzyldithiocarbamate)

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Sep 2021 to 03 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

Adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of stock solutions prepared for each treatment group were collected for analysis at each sampling interval during the test in order to confirm concentrations being delivered to the diluter system. Test solution samples were collected from one replicate test chamber of each treatment and control group 10, 9, 8, 7 and 2 days prior to the start of exposure to confirm concentrations after conditioning the diluter system for 3, 4, 5, 6, 11 days, respectively. Test solution samples also were collected from one replicate test chamber in each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test to measure concentrations of the test substance.

Test solution samples (40 mL) were collected from mid-depth using preconditioned glass graduated pipettes, placed in glass vials, and processed immediately for analysis. Stock solution samples (~2 mL) were collected directly from the tips of the plastic syringes delivering stock solutions in the test system, placed in glass vials, and processed immediately for analysis. Test solution and stock solution samples collected at the first pretest interval were not placed into preconditioned glass vials. Test solution and stock solution samples collected at the second pretest interval were placed into glass vials that were rinsed with the appropriate concentration of solution prior to sampling. Test solution and stock solution samples collected at the third pretest interval and all subsequent analytical intervals throughout the test were placed in glass vials that had been conditioned with the appropriate concentration of solution for one or three days prior to sampling. The OECD No. 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures recommends “preconditioning of test vessels using solutions of the test substance” for adsorbing substances, and states “the concentration of the test substance used to condition a vessel should not exceed the test concentration appropriate to the vessel, otherwise the test substance may desorb during the test and increase the exposure concentration”. This procedure was followed for both the sampling vessels and the stock preparation glassware, in that the same concentration of a solution was used to pre-condition the vessel/glassware.

Vehicle:

yes

Remarks:

0.1 mL/L HPLC-grade dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual stock solutions were prepared for each of the five concentrations tested. Stock solutions were prepared every one or two days in the pretest period, while stock solutions were prepared daily during the exposure period of the test. All test solution concentrations were adjusted to 100% active ingredient during preparation, based on the reported test substance purity (99.8%). At each preparation, a primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 100 μg/mL. The primary stock solution was sonicated for approximately 10 to 30 minutes and inverted to mix. Four secondary stock solutions were prepared in DMF at nominal concentrations of 6.3, 13, 25 and 50 μg/mL by proportional dilution of the primary stock. Beginning with the stock solutions prepared eight days prior to test initiation, the primary stock was stirred on a stir-plate while aliquots were removed for the proportional dilution of the secondary stocks. The secondary stock solutions were inverted to mix.
- Storage of stock solutions: During the pretest period, secondary stock solutions not immediately placed on the test system for delivery were stored under refrigerated conditions. During the exposure period, secondary stock solutions were placed on the test system for delivery immediately after preparation.
- Evidence of undissolved material: After preparation, the primary stock solution and the secondary stock solutions appeared clear and colorless, with no visible signs of precipitate.
- Application: The stock solutions were delivered to the diluter mixing chambers (at a rate of 39.0 L/minute) where they were mixed with dilution water (at a rate of 390 mL/minute) to achieve the desired test concentrations.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Fathead minnow embryos used in the test were obtained from cultures maintained by the test facility.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
The brood fish cultured at the test facility were acclimated for at least 14 days prior to the collection of embryos for the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 24.7 to 26.3ºC, measured with a hand-held digital thermometer. The pH of the water ranged from 8.2 to 8.3, and dissolved oxygen concentrations were ≥7.5 mg/L (≥91% of air-saturation). During the 7 days immediately preceding the test, the brood fish used for the test showed no signs of disease or stress and there was no mortality.
Prior to test initiation, the embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development. The embryonic stage of development at test initiation, using a representative sample of the embryos from the available spawns used to initiate the test, was approximately from early gastrula to three-quarter epiboly.

POST-HATCH FEEDING
- Feeding: Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp., <24 hours old) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Fish were not fed for at least 24 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

4-Day Hatch and 28-Day Post-Hatch

Hardness:

144 - 152 mg/L as CaCO3

Test temperature:

24.4 - 24.9°C

pH:

7.8 – 8.1

Dissolved oxygen:

6.4 - 8.2 mg O2/L
- ≥ 78% ASV

Conductivity:

322 - 442 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 0 (negative control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0 and 10 μg/L
Mean measured concentrations: < LOD, < LOD, 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L, respectively. See Table 1 and Table 2 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Emybro cups: Glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant
- Test vessel: 9-L glass aquaria filled with approximately 7.5 L of test solution
- Fill volume: The depth of the test water in a representative test chamber was approximately 15.7 cm.
- Type of flow-through: Proportional diluter
- Aeration: No
- No. of fertilized eggs/embryos per cup: 20
- No of cup/ treatment and control vessel: 1
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.012 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.246 g fish/L.

TEST MEDIUM
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the test facility site. The well water was passed through a sand filter and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.

WATER PARAMETERS
- Temperature: Temperature was measured in each test chamber at the beginning of the test, weekly during the test and at the end of the test. Water temperature also was monitored continuously during the test in one negative control test chamber. The system measurements were calibrated prior to exposure initiation and verified or recalibrated approximately weekly during the test with a digital thermometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, approximately weekly during the test and at the end of the test.
- Hardness, alkalinity and conductivity: Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, approximately weekly during the test and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness (with 30-minute transition period)
- Light intensity: 611 lux

EFFECT PARAMETERS MEASURED
During the first day of exposure, embryos were observed twice for mortality and eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. On Day 4 of the test, when hatching reached > 90% in the control groups, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, post-hatch and overall survival and growth at test termination were evaluated.
Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60˚C for approximately 46 hours to obtain dry weight data.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

7.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: hatching success, post-hatch larval survival and growth

Details on results:

An overview of the analytical results is provided in Table 1 – Table 2 in ‘Any other information on materials and methods incl. tables’.

- Stock solutions: Analysis of dosing stock solution samples collected during the exposure period yielded measured recoveries that ranged from 60.3 to 109% of the nominal stock concentrations.
- Test solutions: Analysis of test solution samples collected during the exposure period yielded measured recoveries that ranged from 53.8 to 89.8% of nominal test concentrations.

When the measured concentrations of test solution samples collected during the exposure period were averaged, the arithmetic mean measured test concentrations were calculated to be 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L, representing 58, 74, 73, 64 and 71% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

An overview of the biological results is provided in Table 3 – Table 6 in ‘Any other information on results incl. tables’.

- Time of Hatch: Fathead minnow embryos began hatching on Day 3 of the test. The majority of fathead minnow embryos in the control and treatment replicates hatched on Day 4 of the test. Hatching reached > 90% in the control groups on Day 4 of the test, at which time the larvae were released to their respective test chambers.
The mean time to hatch in the negative and solvent control groups was 3.9 and 3.9 days, respectively. There was not a statistically significant difference in the mean time to hatch between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. The mean time to hatch in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 3.9, 3.9, 3.9, 3.9, 3.8 and 4.0 days, respectively. According to Dunnett’s one-tailed test, there were no statistically significant increases or decreases in any treatment group in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for time to hatch were determined to be 7.1 μg/L and >7.1 μg/L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the EC10 and EC20 values for mean time to hatch were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).
- Hatching Success: Hatching success in the negative and solvent control groups was 93.8 and 100%, respectively. There was a statistically significant difference in hatching success between the negative and solvent control groups (p ≤ 0.05). Therefore, the treatment group data were compared to the solvent control alone. Hatching success in the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 98.8, 97.5, 95.0, 98.8 and 95.0%, respectively. According to Fisher’s Exact test, there were no statistically significant decreases evident in hatching success in any treatment group, in comparison to the solvent control (p > 0.05). Consequently, the NOEC and LOEC for hatching success were determined to be 7.1 μg/L and >7.1 μg/L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the solvent control, the LC10 and LC20 values for hatching success were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).
- Post-Hatch and Overall Survival: At test termination, post-hatch larval survival in the negative and solvent control groups was 93.3 and 92.5%, respectively. There were no statistically significant differences in post-hatch larval survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Post-hatch larval survival in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 92.9, 94.9, 88.5, 94.7, 88.6 and 92.1%, respectively. According to Fisher’s Exact test, there were no statistically significant decreases evident in post-hatch larval survival in any treatment group, in comparison to the pooled control (p > 0.05).
Overall survival in the negative and solvent control groups was 87.5 and 92.5%, respectively. There were no statistically significant differences in overall survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Overall survival at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 90.0, 93.8, 86.3, 90.0, 87.5 and 87.5%, respectively. According to Fisher’s Exact test, there were no statistically significant decreases evident in overall survival in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for both post-hatch survival and overall survival were determined to be 7.1 μg/L and >7.1 μg/L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the LC10 and LC20 values for post-hatch larval survival and overall survival were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).
- Biological Observations: In general, the majority of the fish in all control and treatment groups appeared normal throughout the test. Observations of unusual behavior or appearance included appearing small, weak, discolored (pale), morphologically deformed (e.g. crooked spine, having an air bubble in abdomen), and lying on the bottom of the tank with little motion other than minor gill movement. However, these observations were infrequent, did not follow a dose-responsive pattern, were comparable in the controls, and therefore, were not considered to be treatment-related.
- Total Length:  Mean total length at test termination in the negative and solvent control groups was 22.1 and 21.8 mm, respectively. There were no statistically significant differences in mean total length between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Mean total length at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 21.9, 22.6, 22.1, 21.7, 21.6 and 21.8 mm, respectively. According to Dunnett’s one-tailed test, there were no statistically significant decreases evident in total length in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for total length were determined to be 7.1 μg/L and >7.1 μg/L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the EC10 and EC20 values for total length were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).
- Wet Weight: Mean wet weight at test termination in the negative and solvent control groups was 86.0 and 87.2 mg, respectively. There were no statistically significant differences in mean wet weight between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Mean wet weight at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 86.6, 90.4, 86.1, 85.5, 82.4 and 85.1 mg, respectively. According to Dunnett’s one- tailed test, there were no statistically significant decreases evident in wet weight in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for wet weight were determined to be 7.1 μg a.i./L and >7.1 μg/L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the EC10 and EC20 values for wet weight were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).
- Dry Weight: Mean dry weight at test termination in the negative and solvent control groups was 17.4 and 16.8 mg, respectively. There were no statistically significant differences in mean dry weight between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Mean dry weight at test termination in the pooled control and 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 17.1, 18.2, 17.4, 17.1, 16.1 and 16.8 mg, respectively. According to Dunnett’s one-tailed test, there were no statistically significant decreases evident in dry weight in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for dry weight were determined to be 7.1 μg/L and >7.1 μg/L, respectively. Since there was a less than 10% inhibition in any treatment group in comparison to the pooled control, the EC10 and EC20 values for dry weight were empirically estimated to be greater than the highest test concentration (>7.1 μg/L).

Reported statistics and error estimates:

Data from the negative and solvent control groups for each parameter were compared using an
appropriate statistical test (e.g. t-test for continuous-variable endpoints and Fisher’s Exact for
discrete-variable endpoints). Since no differences were detected between the two control groups (p > 0.05) for the mean time to hatch, post-hatch survival, overall survival and growth endpoints, the control data for these parameters were pooled for comparison among the treatment groups. There was a statistically significant difference between the two control groups (p ≤ 0.05) for the hatching success endpoint; therefore, the treatment group data were compared to the solvent control alone.
Hatching success and survival data were considered to be discrete-variable data, while time to hatch and growth data were considered continuous variable data. Discrete-variable data were analyzed using Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (p ≤ 0.05) from the appropriate control group. Continuous variable data were visually examined to determine whether the concentration-response was fundamentally monotonic (trending in one direction, e.g., response not trending up and then down as concentration increases) or non-monotonic. Monotonicity of the dose-response for each endpoint was determined by visual assessment of the data. Since none of the data for the continuous-variable endpoints followed a monotonic concentration response, the step-down Jonckheere-Terpstra trend test was not used to statistically evaluate the data. All continuous-variable data were also evaluated for normality using Shapiro-Wilk’s test, and for homogeneity of variance using Levene’s test (α = 0.01). The data for all continuous-variable endpoints passed the assumptions of normality and homogeneity of variance. Therefore, those treatment means that were significantly different from the appropriate control means were identified using Dunnett’s one-tailed test.

Table 3, Summary of Time to Hatch, Hatching Success, Post-Hatch Larval Survival and Overall Survival of Fathead Minnows Exposed to the test substance

Mean Measured Concentration (µg/L)	Number Exposed	Total Number Hatched	Hatching Success (%)a	Mean Time to Hatch ± Std. Dev. (Days)b	Number Surviving to Termination	Post-Hatch Survival (%)c	Overall Survival (%)c
Negative Control	80	75	93.8	3.9 ± 0.0634	70	93.3	87.5
Solvent Control	80	80	100	3.9 ± 0.222	74	92.5	92.5
Pooled Control	160	155	--d	3.9 ± 0.151	144	92.9	90.0
0.37	80	79	98.8	3.9 ± 0.144	75	94.9	93.8
0.96	80	78	97.5	3.9 ± 0.0455	69	88.5	86.3
1.8	80	76	95.0	3.9 ± 0.134	72	94.7	90.0
3.2	80	79	98.8	3.8 ± 0.225	70	88.6	87.5
7.1	80	76	95.0	4.0 ± 0.101	70	92.1	87.5

a There were no statistically significant decreases evident in any treatment group in comparison to either the negative or solvent control, according to Fisher’s Exact test (p > 0.05).

b There were no statistically significant increases or decreases evident in mean time to hatch in any treatment group in comparison to the pooled control, according to Dunnett’s one-tailed test (p > 0.05).

c There were no statistically significant decreases evident in any treatment group in comparison to the pooled control, according to Fisher’s Exact test (p > 0.05).

d The negative control and solvent control were found to be statistically different for the hatching success endpoint (p ≤ 0.05); therefore, the treatment data were compared to the negative control and solvent control groups separately.

Table 4, Summary of Larval Growth of Fathead Minnows Exposed to the test substance

Mean Measured Concentration (µg/L)	Mean Total Length ± Std. Dev. (mm)a.	Mean Wet Weight ± Std. Dev. (mg)a.	Mean Dry Weight ± Std. Dev. (mg)a.
Negative Control	22.1 ± 0.315	86.0 ± 4.85	17.4 ± 1.19
Solvent Control	21.8 ± 0.693	87.2 ± 6.84	16.8 ± 1.31
Pooled Control	21.9 ± 0.530	86.6 ± 5.52	17.1 ± 1.20
0.37	22.6 ± 0.351	90.4 ± 3.26	18.2 ± 0.905
0.96	22.1 ± 0.292	86.1 ± 4.49	17.4 ± 0.786
1.8	21.7 ± 0.420	85.5 ± 2.39	17.1 ± 0.643
3.2	21.6 ± 0.247	82.4 ± 3.43	16.1 ± 0.408
7.1	21.8 ± 0.351	85.1 ± 4.40	16.8 ± 0.405

a. According to Dunnett’s one-tailed test, there were no statistically significant decreases evident in any treatment group in comparison to the pooled control (p > 0.05).

Table 5, Percent Inhibition as Compared to the Control Groups

Percent Inhibition (%) Compared to the Control Groups a.
Mean Measured Concentration (µg/L)	Hatching Success		Mean Time to Hatch	Post-Hatch Larval Survival	Overall Survival	Mean Total Length	Mean Wet Weight	Mean Dry Weight
	NC	SC	PC	PC	PC	PC	PC	PC
0.37	-5.3	1.2	0.0	-2.2	-4.2	-3.2	-4.4	-6.4
0.96	-3.9	2.5	0.0	4.7	4.1	-0.9	0.6	-1.8
1.8	-1.3	5.0	0.0	-1.9	0.0	0.9	1.3	0.0
3.2	-5.3	1.2	2.6	4.6	2.8	1.4	4.8	5.8
7.1	-1.3	5.0	-2.6	0.9	2.8	0.5	1.7	1.8

PC = compared to the pooled control group; NC = compared to the negative control group; SC = compared to the solvent control group.

a. Stimulation or a greater response in the test substance treatment than the appropriate control is reported as a negative percent inhibition. Calculated using Excel 2010; manual calculations may differ slightly.

Table 6, Effect values

Endpoint	LC/EC10                                                                             (µg/L)	LC/EC20 (µg/L)	NOEC (µg/L)	LOEC (µg/L)
Hatching Success	>7.1	>7.1	7.1	>7.1
Time to Hatch	>7.1	>7.1	7.1	>7.1
Post-Hatch Larval Survival	>7.1	>7.1	7.1	>7.1
Overall Survival	>7.1	>7.1	7.1	>7.1
Total Length	>7.1	>7.1	7.1	>7.1
Wet Weight	>7.1	>7.1	7.1	>7.1
Dry Weight	>7.1	>7.1	7.1	>7.1

 

Validity criteria

The following criteria were used to judge the validity of the test and were met:
1) Dissolved oxygen concentrations remained ≥78% of air saturation (≥6.4 mg/L) throughout the test. (Should be > 60% of the air saturation value throughout the test)

2) The water temperatures manually measured in the test chambers ranged from 24.4 to 24.9°C during the study, while temperature monitored continuously in one replicate of the negative control ranged from 23.48 to 25.28°C throughout the test. (should not differ by more than ± 1.5°C between test chambers or between successive days at any time during the test and should be within the 25 ± 1°C range specified for the test species)
3)The coefficient of variation of the measured concentrations during the study did not exceed 14.3%. (Should be maintained within ± 20% of the mean measured value)
4) Hatching success in the negative and solvent control groups was 93.8 and 100%, respectively, while post-hatch larval survival at test termination in the negative and solvent control groups was 93.3 and 92.5%, respectively. (Should be >70% hatching success, and >75% post-hatch larval survival).

Thus, the study is considered valid.

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In the early life stage toxicity test according to OECD TG 210 and EPA 850.1400 guideline, the 32-day NOEC for hatching success, post-hatch larval survival and growth in fathead minnow was determined to be 7.1 μg/L, based on mean measured concentrations.

Executive summary:

An early-life stage toxicity test with the test item was performed on fathead minnow (Pimephales promelas). The study was performed according to OECD TG 210 and EPA 850.1400 guideline and in compliance with GLP criteria. The embryos (20 embryos/test vessel; 4 vessels/ concentration or controls) were exposed for 32 days (4-day hatch and 28-day post-hatch) to a range of nominal concentrations: 0 (blank control), 0 (solvent control, DMF), 0.63, 1.3, 2.5, 5.0 and 10 μg/L. The mean measured concentrations were < LOD, < LOD, 0.37, 0.96, 1.8, 3.2 and 7.1 µg/L, respectively. The test conditions were: temperature 24.4 – 24.9 ˚C, pH 7.8 – 8.1, dissolved oxygen 6.4 – 8.2 mg/L and light intensity 611 lux (16/8 hours light/dark cycle with 30-minute transition period).

Hatching reached > 90% in the control groups on Day 4 of the test. There was no statistically significant difference in the mean time to hatch between the negative and solvent control groups (p > 0.05). Hatching success in the negative and solvent control groups was 93.8 and 100%, respectively. Hatching success in the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 98.8, 97.5, 95.0, 98.8 and 95.0%, respectively. At test termination, post-hatch larval survival in the negative and solvent control groups was 93.3 and 92.5%, respectively. There were no statistically significant differences in post-hatch larval survival between the negative and solvent control groups (p > 0.05). Post-hatch larval survival in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 92.9, 94.9, 88.5, 94.7, 88.6 and 92.1%, respectively. Overall survival in the negative and solvent control groups was 87.5 and 92.5%, respectively. Overall survival at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 90.0, 93.8, 86.3, 90.0, 87.5 and 87.5%, respectively. The majority of the fish in all control and treatment groups appeared normal throughout the test. Some observed unusual behaviour or appearance were infrequent, did not follow a dose-responsive pattern, were comparable in the controls, and therefore, were not considered to be treatment-related. Mean total length at test termination in the negative and solvent control groups was 22.1 and 21.8 mm, respectively. Mean total length at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 21.9, 22.6, 22.1, 21.7, 21.6 and 21.8 mm, respectively. Mean wet weight at test termination in the negative and solvent control groups was 86.0 and 87.2 mg, respectively. Mean wet weight at test termination in the pooled control and the 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 86.6, 90.4, 86.1, 85.5, 82.4 and 85.1 mg, respectively. Mean dry weight at test termination in the negative and solvent control groups was 17.4 and 16.8 mg, respectively. Mean dry weight at test termination in the pooled control and 0.37, 0.96, 1.8, 3.2 and 7.1 μg/L treatment groups was 17.1, 18.2, 17.4, 17.1, 16.1 and 16.8 mg, respectively. Based on these findings, the NOEC was determined to be the highest concentration tested, 7.1 µg/L (mean measured concentration) for hatching success, post-hatch larval survival and growth.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Minor resitricitons in design and/or reporting but otherwise adequate for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The dilution water was a synthetic medium, Dutch Standard Water according to NPR 6507, containing 100 mg Na HCO3/l, 20 mg KHCO3/l, 200 mg CaCl2.2H2O/l and 180 mg MgSO4. 7H2O/l. The pH is approximately 8.2, the hardness 13 dH. The medium was aerated before use.

Test media of the water-soluble test stubstances were prepared by dissolving the test substance in the dilution water. For the poorly water-soluble substances, a stock solution in acetone or DMSO was made, from which a second stock solution in dilution water was prepared. As the water solubility of most test substances was unknown, the calculations are based on nominal concentrations. The test substances did not always solve well in the dilution water and formed dispersions. Where nominal concentrations exceed the water saturation values, the nominal cocnetrations should be interpreted accordingly. The pH, oxygen concentration and temperature were measured and adjusted if necessary, before the test animals were introduced in the medium.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Brachydanio rerio (new name Danio rerio) were obtained form a local aquarium retailer and kept in the laboratory at 24 ˚C, males and females in separate groups. They were bred at regular intervals: in the afternoon females were isolated in small tanks filled with a mesh bottom at 26 ˚C. After a few hours two males were introduced before the lights went off. In hte morning after the lights were turned on, eggs were produced. Each batch of eggs was collected from the bottom of the tank by a small fishing net and transferred to a small volume of water. White and opaque eggs and empty shells were removed and the quality of the batch and their stage of development was assessed. For each test eggs of only one female were used to insure a more homogeneous population of test eggs. On several occassions, test were repeated, enabling to compare the sensitivity of batches of different origin.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Post exposure observation period:

none

Hardness:

200 mg CaCO3/l

Test temperature:

not specified in report

pH:

8.2

Dissolved oxygen:

saturated

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentrations: 0.1, 0.32, 1, 3.2, 10 mg/l

Details on test conditions:

Test containers were polystyrene multi-dishes with 6 holes each containing 5 ml of test mediu. Five to 7 eggs were transferred to each hole by means of a glass tube equiped with a rubber bulb. CS2- solutions were tested in glass tubes with air-tight teflon-tapered screw caps. The eggs were introduced in the tubes in 5 ml of dilution medium before 25 ml of CS2-solution was added to reach the final concentrations. Incubation started within 4 hours after spawning (gastrula stage). The eggs were incubated at 25 C under artificial light during working hours. Daily observations were made on survival of eggs and larvae, hatching and malformations. Dead organisms were removed. The test was semi-static: the medium was renewed after two or three days. Oxygen concentration, pH and temperature were measured to confirm that test conditions were normal. The NOEC concentrations for survival, hatching and malformations were determined graphically.

Reference substance (positive control):

no

Key result

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: malformations

Results with reference substance (positive control):

not applicable

Description of key information

All available data was assessed. The study representing the worst-case effects is included here and its effect value is used as the key value. Another study is included as supporting information.

Freshwater, 32-d NOEC = 7.1 μg/L (based on mean measured concentrations), hatching success, post-hatch larval survival and growth, Pimephales promelas, OECD TG 210 and EPA 850.1400, Milligan et al. 2022

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

7.1 µg/L

Additional information

There are two studies available for this endpoint. The fish early life-stage toxicity study (Milligan et al. 2022, OECD TG 210 and EPA 850.1400, Reliability 1) was selected as the key study, because it has the highest reliability and represents the worst-case effects (i.e. showed lower NOEC value). The study was performed on fathead minnow (Pimephales promelas). The embryos (20 embryos/test vessel; 4 vessels/ concentration or controls) were exposed to a range of nominal concentrations of 0 (blank control), 0 (solvent control, DMF), 0.63, 1.3, 2.5, 5.0 and 10 μg/L for 32 days (4-day hatch and 28-day post-hatch). The mean measured concentrations were < LOD, < LOD, 0.37, 0.96, 1.8, 3.2 and 7.1 µg/L, respectively. The test conditions were: temperature 24.4 – 24.9 ˚C, pH 7.8 – 8.1, dissolved oxygen 6.4 – 8.2 mg/L and light intensity 611 lux (16/8 hours light/dark cycle with 30-minute transition period). There were no treatment-related effects observed at mean measured concentrations ≤7.1 μg/L. Based on the findings, the NOEC was determined to be 7.1 µg/L (mean measured concentration) for hatching success, post-hatch larval survival and growth. 

Another long-term toxicity study with freshwater fish Danio rerio (tested as Brachydanio rerio) is used as supporting information for this endpoint. The study is a semi-static non-GLP test, which followed a protocol similar to OECD TG 210 (Akzo Nobel Research Laboratories 1991, Reliability 2). The test organisms were exposed to the test substance at nominal concentrations of 0.1, 0.32, 1, 3.2, 10 mg/L. There was no analytical monitoring during the test. After 8 days of exposure, the NOEC was determined to be 100 µg/L for hatched number. The NOEC was > 10 mg/L for mortality and malformations.