Registration Dossier

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jan 2019 to 01 Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
“Fish, Early-life Stage Toxicity Test”, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals
Version / remarks:
OECD series on testing and assessment number 23, 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Slightly yellow powder
Purity/Composition: 96.4116%
Test item storage: At room temperature protected from light

Analytical monitoring:
yes
Details on sampling:
Sampling:
Frequency: One day before the start of exposure to check the functioning of the system (samples were taken from the test concentrations only). At the start, after 7, 14, 21, 28 and 32 days of exposure.
Volume: 1.8 mL
Storage: In case samples were not analysed on the day of sampling, they were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Additionally, reserve samples of 1.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
Preparation of Test Solutions:
The batch of Omnirad 379 tested was a slightly yellow powder with a purity of 96.416%. Weighing of test item and preparation of stock solutions was performed under dimmed light. Stock solutions were kept in the dark. No correction was made for the purity/composition of the test item.

Range-Finding Test:
Stock solutions of 5,000 µg/mL were prepared in dimethylformamide (DMF; Merck, Darmstadt, Germany) daily. Lower stock solutions, i.e. 50 and 500 µg/mL were prepared by diluting the highest stock with DMF. All stock solutions were clear.
Any residual volumes were discarded.

Final Test:
Stock solutions of 5,000 µg/mL were prepared in DMF three times a week. Lower stock solutions, i.e. 500, 900, 1,600 and 2,800 µg/mL were prepared by diluting the highest stock with DMF. All stock solutions were clear. The two highest stock solutions were observed to be slightly yellow, while the lower stock solutions were clear.
Any residual volumes were discarded.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Holding of the Brood Stock:
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, temperature was measured before transferring the parental fish to the breeding system.
Water quality parameters: Will be kept within the optimum limits for the respective fish species.
Ratio male/female: 1:2
Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands).
Time of fertilisation: Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
32 d
Hardness:
>140 mg/L
Test temperature:
25 ± 1.5°C
pH:
6.0-8.5
Dissolved oxygen:
> 60% (i.e. >5.0 mg/L) of oxygen saturation at 25°C
Nominal and measured concentrations:
Target conc. (µg/L) 50, 90, 160, 280, 500

Average exposure conc. (µg/L) 31, 43, 82, 130, 234

Details on test conditions:
Test type: Flow-through, with continuous renewal of test media
Test duration: 32 days
Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
Test vessels: Stainless steel vessels (~1.7 L).
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Experimental design: The experiment started on nominal day 0 with 80 fresh and healthy fathead minnow embryos per test group. The embryos were randomly distributed and divided equally over four stainless steel vessels.
Light period: 16 h photo-period daily.
Feeding: Embryonic phase: no feeding.
Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48-hours old. On day 21 a mixture of Brine Shrimp Nauplii 48-hours and pelleted fish food ᴓ 150 µm (Gemma micro, Skretting, France). Food was supplied ad libitum.
Reference substance (positive control):
no
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 234 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Embryonic surivival
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 234 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: post-hatch survival (larval survival)
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
31 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
31 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length

Range-Finding Test

The results of the range-finding test are presented in Table 2. No mortality or significant clinical effects were observed at any of the test concentrations or the control during the 7-day test period. Hatching started on Day 4 and was completed at Day 5 for all concentrations and the control.

Samples taken from the stock solutions showed that the measured concentrations were generally in agreement with target (77-111%) and remained stable during a 1-Day and a 3-Day period. See Table 3 in Appendix 9. Samples taken from the mixing vessels at the start of the test showed that the concentrations were low for the two highest concentrations (18-46% of target) and in agreement with target at the lowest concentration, i.e. 113%. See Table 4 in Appendix 9. Finally, samples taken from the test concentrations at Day -1 (to check the flow-through system), Day 0 and Day 3 showed that the actual test concentrations, although below target, were stable. See Table 5 in Appendix 9.

All test conditions were maintained within the limits prescribed by the study plan, except for the temperature at the start in one replicate vessel each of the two highest test concentrations. See also Appendix 10.

Table 2       

Survival of Fish Embryos and Larvae during the Range-Finding Test

Target conc. Omnirad 379 (µg/L) No. of embryos Number of survivors (at nominal day) Total survival (%)
Day 0 1 2 3 4 5 6 7
Solvent Control 10 10 10 10 10 10 10 10 100
10 10 10 10 10 10 10 10 100
5.0 10 10 10 9 9 9 9 9 90
10 10 10 10 10 10 10 10 100
50 10 9 9 9 9 9 9 9 90
10 10 10 10 10 10 10 10 100
500 10 10 10 10 10 10 10 10 100
10 10 10 10 10 10 10 10 100

Early Life Stage Test

Measured Concentrations

The results of the analysis of the samples taken during the test period are described in Table 6 of Appendix 9.

Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were below target but relatively stable. Effect parameters were based on the average exposure concentrations. See Table 3 for the calculated exposure concentrations.

Table 3       

Target, Measured and Average Concentrations

Target conc. Omnirad 379 (µg/L) Measured concentration (µg/L) Average exposure conc. (µg/L)
Day 0 Day 7 Day 14 Day 21 Day 28 Day 32
50 25.2 31.0 35.4 31.5 32.8 32.7 31
90 18.0 20.7 56.5 52.2 58.3 51.2 43
160 52.0 78.4 94.5 87.7 93.5 84.6 82
280 77.4 140 154 130 151 129 130
500 127 245 323 224 262 224 234

Embryonic Survival

Figure 1 and Table 4 show the mean data for hatchability (embryonic survival) during the first 8 days of exposure for each of the test groups. Appendix 1, Table 9 lists the survival data per replicate throughout the test period.

The two control groups were compared for hatchability and combined for statistical analysis since no significant difference was found between the control groups, see also Appendix 4. The overall survival of embryos at the end of hatching was 99% in the pooled control, which complies with the requirements of the guideline (i.e. at least 70% of the embryos in the control should hatch). Hatching success was 98-100% in concentrations up to and including the highest average exposure concentration of 234 µg/L and was therefore not statistically different from the pooled control, see also Appendix 4.

Hatching of embryos started and ended between Day 4 and Day 8 of exposure for the control groups and all test concentrations.

Table 4       

Percentage of Embryonic Survival at the End of Hatching

Treatm.[µg/L] Total Introduced Hatched Not Hatched % Hatched
Pooled Control 160 158 2 99
31 80 78 2 98
43 80 79 1 99
82 80 80 0 100
130 80 79 1 99
234 80 80 0 100

Larval Survival and Development

Figure 2 and Table 5 show the mean data for post-hatch survival at the end of the test for each of the test groups. Appendix 1, Table 10 lists data on the post-hatch survival of larvae per replicate.

The two control groups were compared for post-hatch survival and combined for statistical analysis since no significant difference was found between the control groups, see also Appendix 5. The mean post-hatch larval survival was 84% in the pooled control. The validity criterion for post-hatch survival of at least 75% was met. The survival rates in the Omnirad 379 treated groups ranged from 72 to 96%.

Statistical analyses indicated that larval survival was not statistically different from the pooled control at test concentrations up to and including 234 µg/L, see also Appendix 5. A number of effects were observed in this study, see Appendix 1, Table 11. Statistical analysis was not performed on abnormalities, but based on the observations made during the test it appears that the severity and incidence of abnormalities was not concentration dependent and comparable with the controls.

Table 5       

Post-Hatch Survival at the End of Exposure

Average concentration Omnirad 379 (µg/L) Total Hatched Survived Dead % Post-hatch survival
Pooled Control 158 132 26 84
31 78 68 10 87
43 79 76 3 96
82 80 68 12 85
130 79 57 22 72
234 80 58 22 73

Effects on Larval Growth

Body weight and body length measured at the end of the test period are summarised in Appendix 2, Table 12 and Appendix 3, Table 13, respectively. Graphical presentations of body length and weight in different test groups are shown in Figure 3 and Figure 4 respectively. Because no statistically significant difference was observed between control treatments, the blank and solvent control groups were pooled, see also Appendix 6 and Appendix 7.

The test substance caused a reduction in body weight of 6% in fish exposed to an average concentration of 31 µg/L and of 21-28% in fish exposed to average concentrations of 43 up to and including 234 µg/L. See also Table 6. A statistically significant reduction of weight was observed at 43 µg/L and higher, see Appendix 6.

The test substance caused a reduction in body length of 2% in fish exposed to an average concentration of 31 µg/L and of 7-10% in fish exposed to average concentrations of 43 up to and including 234 µg/L. See also Table 7. A statistically significant reduction of length was observed at 43 µg/L and higher, see Appendix 7.

Table 6       

Mean Body Weight (mg) and Percentage of Reduction at the End of Exposure

Average concentration Omnirad 379 (µg/L) Mean Std. Dev. n %Reduction
Pooled Control 76.649 7.5905 8
31 72.208 6.3593 4 5.8
43 58.628 3.8741 4 23.5*
82 60.297 3.6583 4 21.3*
130 60.656 10.4219 4 20.9*
234 54.953 5.8952 4 28.3*

*Effect was statistically significant (p ≤0.05)

Table 7       

Mean body length (mm) and percentage of reduction at the end of exposure

Average concentration Omnirad 379 (µg/L) Mean Std. Dev. n %Reduction
Pooled Control 21.01 0.467 8
31 20.7 0.383 4 1.5
43 19.45 0.436 4 7.4*
82 19.45 0.173 4 7.4*
130 19.35 0.940 4 7.9*
234 18.97 0.538 4 9.7*

*Effect was statistically significant (p ≤0.05)

Experimental Conditions

Experimental conditions are summarized in Table 8.

The individual results are presented in:

•       pH: Appendix 8, Table 14. The pH was within the range described in the study plan (6.0-8.5).

•       Oxygen concentration: Appendix 8, Table 15. Oxygen concentrations was generally > 60% (i.e. >5.0 mg/L) of oxygen saturation at 25°C as prescribed by the study plan, with three exceptions where concentrations of 4.6 and 4.9 mg/L were measured. See also Appendix 10.

•       Temperature: Appendix 8, Table 16. The average temperature measured at weekly intervals in the controls and the treatment groups and the temperature continuously measured in one of the replicates of the control was within the range described in the study plan: 25 ± 1.5°C.

•       Hardness: Appendix 8, Table 17. Total hardness was >140 mg/L, as prescribed by the study plan.

•       The concentration of TOC in sample taken from the adjusted ISO-medium was below the detection limit of the device and as such complied with the value recommended by the guideline (i.e. <2 mg TOC/L).

•       The light intensity during the 16-hour daily light period did not exceed 1,000-1,500 lux, as prescribed by the study plan.

Table 8       

Summary of Experimental Conditions

Condition Range
pH 7.1 - 7.7
Dissolved oxygen concentration (mg O2/L) 4.6 - 9.5
Temperature measured in test vessels (°C) 23.7 - 25.3
Temperature measured continuously in control vessel (°C) 24.1 - 24.9
Hardness (mg CaCO3) 196 - 214
Total Dissolved Organic Carbon content (mg TOC/L) n.d.
Light intensity (Lux) 406 - 763

n.d.: not detectable; concentration was below the detection limit of the method

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the present study assessed the possible lethal and sub-lethal effects of Omnirad 379 during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions:
1. Omnirad 379 did not affect the hatching success (embryonic survival) at concentrations up to and including 234 μg/L (NOEC). Hence the LOEC was >234 μg/L.
2. Omnirad 379 did not affect post-hatch survival (larval survival) at concentrations up to and including 234 μg/L (NOEC). Hence the LOEC was >234 μg/L.
3. Omnirad 379 had a significant effect on the growth of the exposed larvae. Both body weight and body length were significantly affected at a concentration of 43 μg/L (LOEC). The NOEC was 31 μg/L for both weight and length.
Executive summary:

The objective of the study was to define the lethal and sub lethal effects of Omnirad 379 on the early life stages of fish in a flow-through system. For this objective the early-life stages of fathead minnow were exposed for 32 days to a range of concentrations of the test item dissolved in water. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations.

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 210, 2013. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2019.

The batch of Omnirad 379 tested was a slightly yellow powder with a purity of 96.416%. Weighing of test item and preparation of stock solutions was performed under dimmed light. Stock solutions were kept in the dark. The test was performed using a flow-through system with target concentrations of 50, 90, 160, 280 and 500 µg/L, which were based on the results of a preceding range-finding test. A blank- and a solvent control were also included.

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions in dimethylformamide (DMF) were prepared three times per week at a factor of 10,000 higher than the target concentrations. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The test was performed with four replicates containing 20 embryos per replicate for each concentration and the control groups. During the embryonic and larval phases, the embryos/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test, the surviving fish were measured and weighed.

Samples for chemical analysis of the actual Omnirad 379 concentrations were taken one day before the start, at the start, at weekly intervals and at the end of the test. Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were below target but relatively stable. Effect parameters were based on the average exposure concentrations that were calculated to be 31, 43, 82, 130 and 234 µg/L/

The study generally met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the present study assessed the possible lethal and sub-lethal effects of Omnirad 379 during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions:

1.       Omnirad 379 did not affect the hatching success (embryonic survival) at concentrations up to and including 234 µg/L (NOEC). Hence the LOEC was >234 µg/L.

2.       Omnirad 379 did not affect post-hatch survival (larval survival) at concentrations up to and including 234 µg/L (NOEC). Hence the LOEC was >234 µg/L.

3.       Omnirad 379 had a significant effect on the growth of the exposed larvae. Both body weight and body length were significantly affected at a concentration of 43 µg/L (LOEC). The NOEC was 31 µg/L for both weight and length.

Description of key information

Study conducted to recognised testing guideline with GLP certification

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
31 µg/L

Additional information