1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

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:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Early-life stages of fish were exposed to the test substance under flow-through conditions. Egg mortality and hatchability, fry growth and survival of the fish were measured to determine the NOEC of the test item to the fish.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling frequency: Analytical samples were taken from the test concentrations and controls on days 0, 8, 16, 22 and 28.
- Sampling procedure: The concentration of test substance is measured in one of each set of duplicate tanks at each concentration every week, alternating between the duplicate tanks from week to week.

Vehicle:

yes

Remarks:

Acetone The carrier control and the highest test concentration had an acetone concentration of 100 mg/L

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A closed-cell pressure operated proportional diluter was utilized to intermittently deliver the five test concentrations, the solvent control and the blank control solutions. A 100 mg/L stock solution of test item prepared in acetone and distilled water was delivered and dispensed to the mixing vessel of the dilutor to obtain the desired range of test solutions. This stock solution was continuously stirred in paired 15-gallon polyproplene carboys and delivered to the dilutor by a peristaltic pump.
The dilutor was calibrated to deliver 100 percent, 49.5 percent, 25.6 percent, 12.5 percent, and 6.3 percent of the test item stock by volume. These were the approximate desired concentrations based on the range-finding toxicity studies and apparent solubility of the test item in water. The dilutor was calibrated to cycle approximately every 20 minutes so as to insure that a greater than 5 volume turnover rate was met for each test aquaria.
For each treatment level and controls, the test solution (or dilution water) was split and delivered to two replicate glass test chambers by glass splitter vessels and tubing.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Fertilized fathead minnow embryos for the early life stage test were obtained from Aquatic Research Organisms in Hampton, New Hampshire on October 29, 1985. These embryos were up to 24 hours old at the time of collection.
Approximately 1,000 embryos were shipped by courier from Hampton, New Hampshire to Fort Collins, Colorado. Immediately after their arrival at ERT in Fort Collins, the embryos were examined, removed from their substrate, and randomly distributed to each test embryo cup. Approximately 20 to 30 embryos had a fungus infestation upon receipt - a below average percentage of infested embryos.

POST-HATCH FEEDING
- Start date: Day 3 of exposure
- Type of feed: Live, newly-hatched (<48 hour old) brine shrimp nauplii
- Frequency of feeding: Two to three times daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Hardness:

20 - 26 mg CaCO3/L

Test temperature:

23.6 - 25.5 °C

pH:

6.80 - 7.25

Dissolved oxygen:

3.90 - 6.6 mg/L

Conductivity:

63 - 100 µmhos/cm2

Nominal and measured concentrations:

Nominal concentrations: 0 (blank control), 0 (carrier control), 6.3, 12.6, 25.6, 49.5 and 100 mg/L.
Mean measured concentrations: <1.0, <1.0, 4.04, 11.08, 17.60, 36.60 and 68.60 mg/L, respectively.
See also Tables 1 - 6 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Embryo cups: 475 mL glass jars with the bottoms replaced with 600 micron mesh netting. In each test chamber, one embryo cup was suspended from a rocker arm apparatus driven by a 2 rpm motor, thus providing a vertical lifting distance of approximately 2.5 cm. Without agitating the organisms too vigorously this arrangement insured that the embryos were always submerged and that test solution regularly flowed into and out of the cup.
- Test vessel: Glass aquaria 20 cm wide and 40 cm long with a drain hole centered 4.4 cm from the bottom on one end with a retained volume of 3.5 litres.
- Type of flow-through: Proportional diluter
- No. of fertilized eggs/embryos per vessel: 65 at the start. This number was reduced to 35 at 48 hours of exposure.
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2

TEST MEDIUM
- Dilution water: Domestic water was used for dilutions and controls in the early life stage test. The Fort Collins domestic water supply of dechlorinated water (approximately 23 ppm hardness as CaCO3 ) was filtered through an in-house battery of five automatic backflush dechlorinating filters and transported to a large holding tank which was heavily aerated. Turnover rate in this tank was approximately 5 days. This "aging" insured adaquate aeration and water quality for this test system.

WATER PARAMETERS
- Temperature: Recorded continuously in one test chamber and measured and recorded daily in all test concentrations
- Dissolved oxygen (D.O.): Measured and recorded at least twice weekly in all test concentrations.
- pH: Measured weekly in the controls and in the high, medium, and lowest treatment levels.
- Alkalinity, hardness and conductivity: Measured weekly in the controls and in the high, medium, and lowest treatment levels.
- Cleaning: Test chambers were cleaned frequently by removing debris with a large pipette. Screens and drain holes in each test chamber were kept free of debris by periodic brushing.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark.
- Light: A combination of cool white and wide spectrum Gro-lux fluorescent tubes.

EFFECT PARAMETERS MEASURED:
- Egg mortality, hatchability and fry survival: On days 1 through 10 and days 15, 20, 21, 27, 28, 30 and 32 of the study, embryos, and live larvae or juveniles were counted and dead embryos and larvae or juveniles were noted and removed.
- Growth: At the end of the test, survivors were pat dried and weighed to the nearest milligram.

POST-HATCH DETAILS
The mesh on the bottom of the embryo cups retained the embryos but allowed most of the newly hatched larvae to escape into the test chamber, while the screen at the end of the test chamber prevented the larvae from escaping via the drain. Newly hatched larvae were removed from the embryo cup and placed in the test chamber proper, when observed.
- Start hatching: Day 3
- End hatching: Day 7

FERTILIZATION SUCCESS STUDY
Upon receipt at ERT, embryos were examined visually. Empty shells, fungused embryos, unfertilized embryos, and opaque embryos were discarded. Sixty-five healthy, fertile embryos with no visible fungus were randomly transferred into each embryo hatching cup. Hatching cups were then randomly placed in the test chambers. At twenty-four hours after being placed into the cups, embryos were examined visually and all dead or fungused embryos were counted and discarded. The embryos were reduced to 35 embryos per test replicate at 48 hours into the early-life-stage study. On Days 1 through 10 and days 15, 20, 21, 27, 28, 30, and 32 of the study, embryos, and live larvae or juveniles were counted and dead embryos and larvae or juveniles were noted and removed.

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

36.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Embryonic survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

68.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Larval survival, length and weight

Duration:

32 d

Dose descriptor:

other: MATC

Effect conc.:

48.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Survival of embryos and hatched larvae, juvenile length and juvenile weight

Details on results:

At the completion of hatch (Day 7), hatching success in the control and carrier control embryos was at least 91 %. Survival at the end of the test was greater than 80 % in both the control and carrier control. One-way ANOVA indicated that there was no significant difference between the dilution control and carrier control groups, and the carrier control was selected as the appropriate control for further statistical testing. In test groups demonstrating replicate homogeneity (two-tailed F(max) tests), data was pooled for ANOVA testing. Replicates in heterogeneous groups (dilution control and the 17.6 mg/L treatment) were tested as a group and replicate data were not pooled.
Carrier control and control fish had a mean dry weight of 0.0370 and 0.0351 g, and a mean total length of 14.95 and 14.80 mm, respectively, at the end of the test. See also Tables 7 and 8 in 'Any other information on results incl. tables'.

Reported statistics and error estimates:

Chi-square analyses of R X C contingency tables (Zar 1974) were applied to analyze the following mortality parameters:
A) healthy, fertile embryos at 48 hours
B) embryos that produced live larvae
C) embryos that produced live juveniles at the end of the test
D) hatched larvae producing live juveniles at the end of the test

At the end of the test all surviving juvenile fish were weighed and measured. For weights and lengths two-tailed F(max)tests (Sokal and Rohlf 1969) were used to determine equality or inequality of replicates. Individual juvenile fish weights and lengths were used as separate data points for one way analyses of variance (ANOVA). Each treatment level was then compared to the appropriate control using a two-tailed Dunnett's test if ANOVA was significant (Zar 1974).

Table 7. Summary of mortality effects

 

Measured Treatment Concentration (mg/L)	% Healthy Fertile Embryos at 48 Hours	% Embryos Producing Live Larvae	% Embryos Producing Live Juveniles at End of Test	% Hatched Larvae Producing Live Juveniles at End of Test
Control	97	100	86	86
Carrier Control	91	100	80	80
4.04	95	99	66	67
11.08	95	100	84	84
17.60	88	100	77	77
36.60	93	96	77	81
68.60	75a	99	76	77

a Significantly different from carrier control group at α = 0.05 (Scheffe’s test)

Table 8. Summary of weight and length at the end of the test

 

Measured Treatment Concentration (mg/L)	Weight (gms)		Length (mm)
	Mean	S.D.	Mean	S.D.
Control	0.0351	0.0155	14.80	0.575
Carrier Control	0.0370	0.0490	14.95	0.587
4.04	0.0394	0.0529	15.41a	0.678
11.08	0.0308	0.0412	13.49a	0.659
17.60	0.0337	0.0447	13.61	0.801
36.60	0.0354	0.0447	13.78	0.671
68.60	0.0355	0.0447	14.96	0.573

a Significantly different from carrier control group at α = 0.05 (Dunnett's test)

Comment

It is noted that guideline, expiry date of test item, validity criteria were not reported. However this was not regarded as having significant impact on the outcome of the study. Thus,the results are considered acceptable.

Validity criteria fulfilled:

not specified

Remarks:

See comments in 'Any other information on results incl. tables'

Conclusions:

In the early life stage toxicity test according to an internal protocol of the test facility, the 32-day NOEC for embryonic survival in fathead minnow was determined to be 36.6 mg/L, based on mean measured concentrations.

Executive summary:

The early-life stage toxicity of the test item was performed on fathead minnow (Pimephales promelas). The study was carried out in accordance with an internal protocol of the test facility. The study was not performed according to GLP criteria. The fertilised eggs (65 eggs/ test vessel; 2 vessels/ concentration or controls) were exposed to a range of nominal concentrations of 0 (blank control), 0 (carrier control), 6.3, 12.5, 25.6, 49.5 and 100.0 mg/L for 32 days. The mean measured concentrations were < 1.0, < 1.0, 4.04, 11.08, 17.60, 36.60 and 68.60 mg/L, respectively. The test conditions were: temperature 23.6 - 25.5 ˚C, pH 6.80 – 7.25, dissolved oxygen 3.90 – 6.6 mg/L and 16 / 8 hours light/dark cycle.

At the completion of hatch (Day 7), hatching success in the control and carrier control embryos was at least 91 %. Fry survival at the end of the test was greater than 80 % in both the control and carrier control. One-way ANOVA indicated that there was no significant difference between the dilution control and carrier control groups, thus the carrier control was selected as the appropriate control for further statistical testing. Carrier control and control fish had a mean dry weight of 0.0370 and 0.0351 g, and a mean total length of 14.95 and 14.80 mm, respectively, at the end of the test. In regards to larval and juvenile mortality, length and weight the treatment levels of the test item were not statistically different when compared to the carrier control group. In regards to embryo mortality, the highest treatment group, 68.6 mg/L was significantly different from the carrier control group. Based on the findings, the NOEC for embryonic survival was determined to be 36.6 mg/L (mean measured concentrations). The NOEC was determined to be 68.6 mg/L for larval survival, length and weight, based on mean measured concentrations. The MAT limit is calculated as 48.01 mg/L.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jul 2014 to 21 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996 draft version

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1241-05

Version / remarks:

2005

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling frequency: Water samples were collected from alternating replicate test chambers of each treatment and control group on Days -2 (to check the flow-through system) , 0, 7, 14, 21, 28 and 34. An additional sample was collected from the 1.3 mg/L test chamber on Day 29 to investigate the high measured concentration on Day 28 of the test.
- Sampling procedure: Samples were collected from mid-depth and placed in glass scintillation vials.
- Sample storage: No, samples were processed immediately for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
Stock solutions were prepared daily during the test. A primary stock solution was prepared in pH adjusted (alkaline) dilution water (filtered saltwater) at a nominal concentration of 801 mg/L. At each stock preparation, three solutions of pH adjusted dilution water were prepared by adding 40 to 140 mL of 0.1 M sodium hydroxide (NaOH) solution into each of the calibrated glass beakers containing 2-L of filtered saltwater and stirred to mix using a stir bar and a stir plate. The final pH of the pH adjusted water ranged from ~8.2 to 9.3. A calculated amount of test substance was weighed into a tared glass beaker and was thoroughly rinsed into 2-L glass volumetric flask using portion of the pH adjusted dilution water. The stock solution was sonicated for approximately 120 minutes followed by inversion to mix. At the end of the mixing, the primary stock solutions appeared clear and colorless. The stock solution was pumped into the diluter mixing chambers assigned to the 0.63 and 1.3 mg/L treatment groups using syringe pumps and to the diluter mixing chambers assigned to 2.5, 5.0 and 10 mg/L treatment groups using a FMI pump, and mixed with dilution water.

APPLICATION OF TEST SOLUTION
A continuous-flow diluter was used to deliver each concentration of the test item and a negative control (dilution water). Syringe pumps and fluid metering pumps were used to deliver volumes of a single test item stock solution to mixing chambers indiscriminately assigned to each treatment. The stock solution was diluted with filtered saltwater in the mixing chambers in order to obtain the desired test concentrations prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at approximately weekly intervals during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately 6.2 volume additions of test solution in each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test. Periodically during the test (i.e. on Friday during the first week and weekly on Tuesday during the remaining of the exposure period), all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Sheepshead minnow embryos used in the test were supplied by Aquatic BioSystems, Inc., Fort Collins, Colorado and were received at Wildlife International free-floating. Upon receipt, the embryos were examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development. Embryos collected for use in the test were approximately 21 to 26 hours old at test initiation. The embryonic stage of development at initiation of the study was between the stages of embryonic shield differentiation and blastopore closure.

POST-HATCH FEEDING
Newly-hatched larvae were fed an excess amount of live brine shrimp nauplii (Artemia sp.) 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. Brine shrimp nauplii were obtained by hatching cysts. Fish were not fed for approximately 48 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:

saltwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

6 days pre-hatching and 28 days post-hatching

Test temperature:

24.5 - 25.5 °C

pH:

7.8 - 8.0

Dissolved oxygen:

6.1 - 7.4 mg O2/L

Salinity:

19 - 22

Reference substance (positive control):

no

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching, larval survival, length, wet weight, dry weight

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching, larval survival, length, wet weight, dry weight

Details on results:

All viable embryos in the control, 0.68, 1.5, 2.6, 5.8 and 10 mg/L treatment replicates started to hatch on Day 5 of the test. Hatching success in the negative control group was 99%. Hatching success in the 0.68, 1.5, 2.6, 5.8 and 10 mg/L treatment groups was 99, 99, 96, 95 and 96%, respectively. Fisher’s Exact test indicated that there were no statistically significant decreases in hatching success in any treatment groups when compared to the negative control (p > 0.05).

Larval survival in the negative control group at test termination was 100%. Larval survival in the 0.68, 1.5, 2.6, 5.8 and 10 mg/L treatment groups was 100, 97, 100, 100 and 99%, respectively. No statistically significant decreases in larval survival were detected in any of the treatment groups when compared to the negative control (Fisher’s Exact test, p > 0.05). The majority of the fish in the negative control, and the 0.68, 1.5, 2.6, 5.8 and 10 mg a.i./L treatment groups appeared normal throughout the test, with occasional observations of fish that were small, weak, observed lying on the bottom of the test chambers or noted with morphological deformity (e.g. curled/curved or crooked spine). Since fish that appeared small were also observed in the negative control and the observations of fish that were weak, deformed or lying on the bottom of the test chambers in the triasulfuron treatment groups were isolated to a few individuals, the sublethal effects noted for the fish in the triasulfuron treatment groups were not considered to be treatment-related.

Statistically significant differences from the control were noted on growth (measured as total length, wet and dry weight) at 0.68, 2.6, 5.8 and 10 mg/L treatment concentrations, but the differences among mean weight and length of fish in the controls and the treatment groups were small while within group variability was very low. Comparisons among such tight groupings of data can be misleading since small differences in means that are not biologically meaningful will be identified as being statistically significant. While Dunnett’s test identifies differences between a single concentration and the control, it does not evaluate the pattern of the response at multiple concentrations. It is a concentration dependent pattern that is a critical in attributing causality of the effect to the test substance. Exposure to increasing concentrations of the test substance would be expected to be associated with increasing response (decrease weight or length). Yet, in this study, the lengths and weights of fish from the lowest concentration group were lower than in the highest concentration group and the means in all treatment groups were greater than the means for historic controls. Thus, although lengths and weights in some treatment groups showed statistically significant differences from the controls when the data were subjected to analysis using Dunnett’s test, it does not appear to be appropriate to interpret the differences as reflecting responses to the test substance.
Under these circumstances, it is appropriate to test whether there is any credible evidence of a concentration related response. The Jonckheere-Terpstra trend test is an appropriate statistical test for the existence of a concentration related trend, and was therefore applied to the length and weights of the fish from this study. According to the Jonckheere-Terpstra test, there is not a significant concentration related trend.

See also Table 4 in 'Any other information on results incl. tables'.

Reported statistics and error estimates:

Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated from the number of larvae that survived to test termination as a percentage of the number of embryos that hatched successfully. Hatching success and survival data were considered to be discrete-variable data, while growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the negative control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test, and for homogeneity of variance using Levene’s tests (α = 0.01). Since the data passed the assumptions of normality and homogeneity of variances, those treatments that were significantly different from the negative control means were identified using Dunnett’s one-tailed test (α = 0.05). In addition, Jonkheere-Terpstra trend test (α = 0.05) was also performed to determine the NOEC for the growth endpoints.

Table 4, Summary of Hatching Success, Larval Survival and Growth

Mean Measured Concentration (mg/L)		Percent Survival to Day 28 Post-Hatch a.	Growth Parameters at Day 28 Post-Hatch
	Percent HatchingSuccess a.		Mean Total Length ± SD (mm) b.	Mean Wet Weight ± SD (mg) b.	Mean Dry Weight ± SD (mg) b.
Negative Control	99	100	20.1 ± 0.28	104.2 ± 3.13	25.1 ± 0.57
0.68	99	100	19.2 ± 0.11*^,∆	92.3 ± 2.40*^,∆	21.7 ± 0.42*^,∆
1.5	99	97	19.7 ± 0.22	104.4 ± 2.88	24.6 ± 0.74
2.6	96	100	19.3 ± 0.28*^,∆	94.5 ± 1.90*^,∆	22.4± 0.50*^,∆
5.8	95	100	19.7 ± 0.24*^,∆	99.3 ± 1.81*^,∆	23.4 ± 0.48*^,∆
10	96	99	19.5 ± 0.14*^,∆	100.0 ± 1.41*^,∆	24.0 ± 0.60*^,∆
EC10 (95% confidence interval) (mg/L)	NA	NA	NA	NA	NA
EC20 (95% confidence interval) (mg/L)	NA	NA	NA	NA	NA
NOEC (mg/L)^†	10	10	10	10	10
a. There were no statistically significant differences in percent hatching success or survival when compared to the negative control (Fisher’s Exact test, p>0.05). b. There were no statistically significant differences in growth when compared to the negative control (Jonkheere-Terpstra Trend test, p>0.05). * Indicates significant differences in growth endpoints from the control (Dunnett’s one-tailed test, p≤0.05). ∆ The statistically significant differences in total length, wet and dry weight found in the 10 mg/L treatment level when compared to the negative control were not considered to be biologically meaningful because the differences were slight (below 5%). Since the mean total length, wet and dry weight in the 0.68, 2.6 and 5.8 mg/L treatment groups did not follow a dose response pattern and were comparable to the historical control data (18.7 ± 1.7 mm for length, 81.9 ± 22.2 mg for wet weight and 18.9 ± 5.9 mg for dry weight), the statistically significant differences noted were not considered to be biologically meaningful. † NOEC values for growth endpoints were determined using Jonkheere-Terpstra Trend Test (p=0.05). NA = not applicable. The ECx values calculated were outside the data used for the calculation and/or the 95% confidence interval were overly wide. Since the results were extrapolation rather that interpolation, the results were not reported.

Validity criteria

1.) the percentages of embryos in the negative control that hatched successfully was 99 %, and the post-hatch survival in the negative control was 100 %. The criteria for validity as outlined by the protocol was > 75 % control hatchability and > 80 % control larval survival;
2.) the dissolved oxygen concentration was > 60 percent of the air saturation value (≥ 6.1 mg/L) throughout the test;
3.) the water temperature measurements did not differ by more than ± 1.5 ºC between test chambers or between successive days at any time during the test, and were within 25 ± 1 °C;
4.) the concentrations of the test substance in solution were satisfactorily maintained within ± 20 % of the mean measured values, with the percent coefficient of variation around the mean ranging from 6.40 to 17.1 %.

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 with sheepshead minnow according to OECD TG 210, EPA 850.1400 (draft) and ASTM Standard E 1241-05 guidelines, the 34-day NOEC for hatching, larval survival and growth was determined to be 10 mg/L, based on mean measured concentrations.

Executive summary:

The early-life stage toxicity of the test item was performed on sheepshead minnow (Cyprinodon variegatus). The study was carried out in accordance with OECD TG 210, EPA 850.1400 (draft) and ASTM Standard E 1241-05 guidelines, and performed according to GLP criteria. Eighty embryos per treatment and control (4 replicates, 20 embryos per replicate) were exposed to a range of nominal concentrations of 0 (blank control), 0.63, 1.3, 2.5, 5.0 and 10 mg/L for 34 days (a 6-day hatching period plus a 28-day post-hatch growth period). The mean measured concentrations were < 1.0, 0.68, 1.5, 2.6, 5.8 and 10 mg/L, respectively. The test conditions were: temperature 24.5 - 25.5 ˚C, pH 7.8 – 8.0, dissolved oxygen 6.1 – 7.4 mg O2/L, salinity 19 - 22 ‰ and a daily photoperiod of 16 hours with a light intensity of 572 lux.

Hatching started on Day 5 in all treatments and the control. Hatching success in the negative control group was 99%. Hatching success in the 0.68, 1.5, 2.6, 5.8 and 10 mg a.i./L treatment groups was 99, 99, 96, 95 and 96%, respectively. There were no statistically significant decreases in hatching success in any treatment groups when compared to the negative control (p > 0.05). Larval survival in the negative control group at test termination was 100%. Larval survival in the 0.68, 1.5, 2.6, 5.8 and 10 mg a.i./L treatment groups was 100, 97, 100, 100 and 99%, respectively. No statistically significant decreases in larval survival were detected in any of the treatment groups when compared to the negative control (Fisher’s Exact test, p > 0.05). Statistically significant differences from the control were noted on growth (measured as total length, wet and dry weight) at 0.68, 2.6, 5.8 and 10 mg/L treatment concentrations, but the differences among mean weight and length of fish in the controls and the treatment groups were small while within group variability was very low. Under these circumstances, it is appropriate to test whether there is any credible evidence of a concentration related response. According to the Jonckheere-Terpstra test, there is not a significant concentration related trend, and when the test is applied in a step-down procedure, the NOEC is found to be 10 mg/L. Based on these results the 34-day NOEC for hatching, larval survival and growth was determined to be 10 mg/L, based on mean measured concentrations.

Description of key information

Freshwater: NOEC 68.6 mg/L for larval survival, length and weight (no guideline followed, Hamaker 1986).

Marine water: 34d NOEC 10 mg/L for hatching, larval survival and growth (OECD TG 210, Minderhout 2015). 

Additional information

Studies required in Annex VII and some other studies required for higher tonnage are available. All the studies available have been included in the dossier for this endpoint. The lowest effect value of these studies was considered in the aquatic toxicity classification and PBT assessment. Therefore, all supporting studies are summarized below. 

There are two studies available for this endpoint, one on freshwater species and one on marine water species. Only the marine water study was performed according to standard guidelines and under GLP.

Freshwater: Fertilised eggs from Pimephales promelas (65 eggs/ test vessel; 2 vessels/ concentration or controls) were exposed to a range of nominal concentrations of 0 (blank control), 0 (carrier control), 6.3, 12.5, 25.6, 49.5 and 100.0 mg/L for 32 days in a flow-through system. The mean measured concentrations were < 1.0, < 1.0, 4.04, 11.08, 17.60, 36.60 and 68.60 mg/L, respectively. The test conditions were: temperature 23.6 - 25.5 ˚C, pH 6.80 – 7.25, dissolved oxygen 3.90 – 6.6 mg/L and 16 / 8 hours light/dark cycle. The NOEC for embryonic survival was determined to be 36.6 mg/L (mean measured concentrations). The NOEC was determined to be 68.6 mg/L for larval survival, length and weight, based on mean measured concentrations. (Hamaker 1986).

Marine water: Fertilised eggs from Cyprinodon variegatus (4 replicates, 20 embryos per replicate) were exposed to a range of nominal concentrations of 0 (blank control), 0.63, 1.3, 2.5, 5.0 and 10 mg/L for 34 days in a flow-through system. The mean measured concentrations were < 1.0, 0.68, 1.5, 2.6, 5.8 and 10 mg/L, respectively. The test conditions were: temperature 24.5 - 25.5 ˚C, pH 7.8 – 8.0, dissolved oxygen 6.1 – 7.4 mg O2/L, salinity 19 - 22 ‰ and a daily photoperiod of 16 hours with a light intensity of 572 lux. The 34- day NOEC for hatching, larval survival and growth was determined to be 10 mg/L, based on mean measured concentrations (Minderhout 2015).