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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
required for a non-EU registration
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
test temperature was too high
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
During the test, the concentrations of the test chemical was analysed on two occasions per week, in fresh and old media (i.e. analyses were made on a sample from the same solution when freshly prepared and at renewal). For determination of test item concentrations in the semi-static test, about 150 mL sample was taken from each test solution and control solution. For sampling of the old test media, one replicate of the four replicates was sampled randomly. 50 mL sample was pretreated to determine the test item concentration, and the remainder was stored in a refrigerator (2-8 °C) for possible re-analysis. The pretreatment method of test samples was identical with that of the recovery test samples.
Vehicle:
no
Details on test solutions:
A saturated solution method: 1.00g (1.0001g~1.0021g) of test item was weighed and directly added into 10L test water, the solution was vigorously blended by magnetic stirring for about 48 hours (±10 minutes). After magnetic stirring at 520~570 rpm, the solution was filtered to remove undissolved test item, and the filtered solution was collected for preparation of diluted solutions and exposure.
At the earlier stage of test, 62.5, 125, 250, 500 mL of the stock solution was diluted to 1000 ml with test water respectively and the solutions were mixed thoroughly; When 1000 ml glass beaker was used as the test chamber, 250, 500, 1000, 2000 mL of the stock solution was diluted to 4000 ml with test water respectively and the solutions were mixed thoroughly.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The animal care and use activities required for conduct of this study were reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of the facility based on Instructive Notions with Respect to Caring for Laboratory Animals (2006, PRC Ministry of Science).
The batch of brood stock was zebra fish 20161128. The brood fish were propagated and cultivated in the laboratory, whose parental generation was introduced from Hangzhou Hunter Biotechnology Inc. and the species of parent fish was AB strain. The brood stock was held in water of the quality to be used in the test during the cultivation period and under the following conditions:
Light condition: 12 hours light: 12 hours darkness;
Temperature: 26 ± 1.5 oC;
Feeding: brine shrimp nauplii (BSN) or flake food twice on weekday and one time on weekend.

Feeding of fish during the test: the time to first feeding was 2 days post hatch. Commercial larvae food for newly-hatched larvae and commercial juvenile food for juveniles twice daily. Live food provides a source of environmental enrichment and therefore should be used in addition to dry food. BSN was used in addition to dry food once daily. Feeding was approximately equal across replicates. Surplus food and faeces were removed as necessary, to avoid accumulation of waste.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d
Remarks on exposure duration:
Hatching was complete by day 4, day 4 was designated day 0 of the post-hatch growth period. The test duration lasted to 30 days post-hatch. The whole test duration was 34 days.
Hardness:
190 mg/L CaCO3
Test temperature:
25.9 - 29.8 degee C, the whole temperature difference was 3.9 degree C, 50% of monitored temperature data exceeded the high limit 27.5 degree C. The temperature didn’t meet requirement. The deviation did not affect the quality or integrity of the study because all control replicates behaved normal and met the validity criteria.
pH:
7.79 - 8.25
Dissolved oxygen:
73.2 - 98.9% of air saturation value
Nominal and measured concentrations:
nominal: 0.144, 0.288, 0.575, 1.15 and 2.3 mg/L
geometric mean: 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass beakers at the earlier stage of the test and 1000 ml glass beakers after 11 days post-hatch
- Material, size, headspace, fill volume: 200 ml test solution was held in 250 ml glass beakers and 1000 ml in 1000 ml glass beakers.
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): semi-static, daily
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Dilution water
Origin: The water prepared using Aquapro Pure Water System
Water hardness: Above 140 mg/L (as CaCO3)
pH: 6.0 to 8.5 pH units, within a range of ± 0.5 pH units during the test
TOC content: < 2 mg/L
Aeration: Before usage, the water was aerated for more than 24 hours to attain dissolved oxygen saturation.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Stage of embryonic development, Hatching and survival, Abnormal appearance, Abnormal behaviour, Weight, Length

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE-FINDING STUDY
- Test concentrations: control, 0.23 and 2.3 mg/L
- Results used to determine the conditions for the definitive study: 0.23 mg/L no significant difference to the control in any of the endpoints. 2.3 mg/L severe effects were found, no fish survived.
Reference substance (positive control):
not specified
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
larvae
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: abnormal appearance of embryos
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: abnormal behaviour or appearance of larvae
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.124 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.124 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: day of 90% hatching
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.124 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: end of hatching
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.345 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
0.345 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: begin of hatch
Details on results:
Survival: The mean of the replicate survival rates for the embryos exposed to the control and the measured concentrations of 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L at hatch were 100%, 100%, 100%, 100%, 90.0% and 36.3%, respectively. At day 34, the survival rate of post-hatch success were 83.8%, 80.0%, 77.5%, 80.0%, 41.4% and 1.6%, respectively.
Sublethal effects:
Morphological and behavioural abnormalities: At the control and the measured concentrations of 0.035, 0.045, 0.073 mg/L, there were no abnormal appearance or behaviour observed on embryos and larvae. At the concentrations of 0.124 and 0.345 mg/L, the abnormal appearances such as curved body and yolk sac oedema were observed on embryos, the abnormal appearances or behaviour such as curved body, pericardial oedema, atypical quiescence and uncoordinated swimming were observed on larvae. The LOEC was 0.124 mg/L.
Time to hatch: Hatch began on day 3 of the test at all groups, hatching was complete by day 4 at the control and the test groups of 0.035, 0.045, 0.073 mg/L, hatching was complete at the group of 0.345 mg/L until day 6.
Effects on dry weight and length:
Mean dry weight per surviving fish for the control and the test groups of 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L was 1.69, 1.67, 1.60, 1.65, 2.88 and 2.32 mg. The increasing weight was observed at the surviving fish at the test groups of 0.124 and 0.345 mg/L.
Mean total length per surviving fish for the control and the test groups of 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L was 9.77, 10.01, 9.70, 9.92, 11.67 and 12.08 mm. The increasing total length was observed at the surviving fish at the test groups of 0.124 and 0.345 mg/L.

Reported statistics and error estimates:
The data were used for statistical analysis using Two-sided Student t-test with a 5% significance level to obtain the lowest observed effect concentration (LOEC). The no observed effect concentration (NOEC) was taken to be the test concentration immediately below the LOEC.
Probit analysis (EPA Version 1.5) was used to calculate LC50 of embryo and larvae and EC50 of abnormal appearance or behaviour of embryo and larvae.

Analytical results: The results showed that the arithmetic mean concentrations in fresh mediawere0.0994, 0.181, 0.353, 0.726 and 1.429 mg/L, which were 61.4% ~ 69.0% of the nominal concentration, the determined concentrations in old media were significantly decreased and many values were lower than LOQ or no signal was detected.The mean exposure concentration over the whole period was calculated from the geometric mean concentration of each renewal period (daily). For the nominal concentrations of 0.144, 0.288, 0.575, 1.15 and 2.3 mg/L, the mean exposure concentrations were 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The study is valid because it meets all validity criteria. The lowest NOEC is 0.073 mg/L measured geometric mean.
Executive summary:

Fish, Early-life Stage ToxicityTestof Ethylene glycol dibenzoate was conducted withzebra fish (Danio rerio) in our facility.

Based on the results of a preliminary test, the definitive test was conducted at 5 nominal concentrations of 0.144, 0.288, 0.575, 1.15 and 2.3 mg/L, concurrently with a blank control group. 80 fertilised eggs were used for test concentrations and control and divided equally between 4 replicate test chambers.

The test was performed with a semi-static system andfrequency of solution renewalwas daily. Thetest organisms were retainedin the beakers whilst 4/5 of test solution/control volume was changed.

During the exposure period, observations on hatching and survival were made one time on 3 hours after exposure and then daily and the number were recorded.For embryos, larvae and juvenile fish,theabnormal appearance or behaviourobserved were also recorded. The dry weightson a replicate basisand individual total lengths were measured at the end of the test.

The concentrations of test item were determined in samples of test media by HPLC method. During the test, the concentrations of the test chemical were analysedon two occasions perweek, in fresh and old media(i.e. analyses were made on a sample from the same solution when freshly prepared and at renewal). The mean exposure concentration over the whole period was calculated from the geometric mean concentration of each renewal period (daily). For the nominal concentrations of 0.144, 0.288, 0.575, 1.15 and 2.3 mg/L, the mean exposure concentrations were 0.035, 0.045, 0.073, 0.124 and 0.345 mg/L.

At the control and measured concentrations of 0.035, 0.045, 0.073 mg/L, there were noabnormal appearance or behaviourobserved onembryos and larvae. At the measured concentrations of 0.124 and 0.345 mg/L, theabnormal appearances such as curved body and yolk sac oedema were observedonembryos,theabnormal appearances or behaviour such as curved body, pericardial oedema, atypical quiescence and uncoordinated swimming were observed on larvae.

Hatching rate and survival rate of post-hatch success, abnormal appearance or behaviourofembryos and larvae,the dry weightson a replicate basisand individual total lengths,days to begin of hatch, 90% hatching and end of hatchingwere calculated. The data wereusedfor statistical analysis usingTwo-sidedStudentt-test with a 5% significance level. Probit analysis (EPA Version 1.5) was used to calculate LC50of embryo and larvae and EC50of abnormal appearance or behaviour of embryo and larvae.

The NOEC/LOEC and LC50/ EC50for biological endpoints are as follows, based onthegeometricmeanconcentration measured:

Biological endpoints

NOEC (mg/L)

LOEC (mg/L)

LC50/EC50and 95% confidence limits (mg/L)

Survival:

Hatching rate (embryo survival)

0.124

0.345

0.278 (0.248~0.317)

Survival rate of post-hatch success (larvae survival)

0.073

0.124

0.131 (0.115~0.149)

Growth at the end of test:

Dry weightson a replicate basis

0.073

0.124

-

Individual total lengths

0.345

>0.345

-

Other observed effects:

Abnormal appearanceofembryos

0.073

0.124

0.164 (0.150~0.181)

Abnormal appearance or behaviouroflarvae

0.073

0.124

0.126 (95% confidence limits was not obtained)

Day to begin of hatch

0.345

>0.345

-

Day of 90% hatching

0.124

0.345

-

Day of end of hatching

0.124

0.345

-

Description of key information

A long-term fish study according to OECD 210 guideline under GLP was conducted with Zebra fish (Danio rerio).

The test was performed with a semi-static system, and test solutions were renewed every 24 hours.

During the exposure period, observations on hatching and survival were made one time on 3 hours after exposure and then daily and the number were recorded. For embryos, larvae and juvenile fish, the abnormal appearance or behaviour observed were also recorded. The dry weights on a replicate basis and individual total lengths were measured at the end of the test.

The geometric mean exposure concentration were calculated and used for endpoint calculations.

The NOEC/LOEC and EC50/LC50for biological endpoints are as follows, based on the geometric mean concentration measured:

Biological endpoints

NOEC (mg/L)

LOEC (mg/L)

EC50/LC50and 95% confidence limits (mg/L)

Survival:

Hatching rate (embryo survival)

0.124

0.345

0.278 (0.248~0.317)

Survival rate of post-hatch success (larvae survival)

0.073

0.124

0.131 (0.115~0.149)

Growth at the end of test:

dry weights on a replicate basis

0.073

0.124

-

individual total lengths

0.345

>0.345

-

Other observed effects:

Abnormal appearance of embryos

0.073

0.124

0.164 (0.150~0.181)

Abnormal appearance or behaviour of larvae

0.073

0.124

0.126 (95% confidence limits was not obtained)

Day to begin of hatch

0.345

>0.345

-

Day of 90% hatching

0.124

0.345

-

Day of end of hatching

0.124

0.345

-

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.073 mg/L

Additional information