Condensation products of Dihydrofuran-2,5-dione, mono-alkenyl derivs. with polyethylene amine and acetic anhydride

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2015 to 29 April 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-700
- Expiration date of the lot/batch: 1 October 2015
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature under nitrogen in the dark
- Stability under test conditions: Stable in container after opening. Stable in water, light and soil.

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

Using a magnetic stirrer, loading rate water-accommodated fractions (WAFs) of 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L and 100 mg/L were prepared by mixing 0.05 g, 0.09 g, 0.16 g, 0.29 g and 0.51 g of test substance in 5000 mL test water respectively. After 24 hours the stirring was stopped and the mixture was left to stand for approximately 1 hour. The WAF solutions were removed by siphoning mid-depth of the mixture, discarding the first 75-100 mL.

Vehicle:

no

Details on test solutions:

Using a magnetic stirrer, loading rate water-accommodated fractions (WAFs) of 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L and 100 mg/L were prepared by mixing 0.05 g, 0.09 g, 0.16 g, 0.29 g and 0.51 g of test substance in 5000 mL test water respectively. After 24 hours the stirring was stopped and the mixture was left to stand for approximately 1 hour. One blank control without test substance was also prepared by this method. The WAF solutions were removed by siphoning mid-depth of the mixture, discarding the first 75-100 mL.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Danio rerio
- Source: Guangzhou Yangshi Breeding Farm
- Weight at study initiation (mean and range, SD): 50-100 mg, individual difference is not more than 25%

FEEDING DURING TEST
Fed twice a day, as much as about 2% body weight, except 24 hours before test initiation.

Test type:

semi-static

Water media type:

other: Dechlorinated tap water

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

The fish were observed daily for external abonormalities and mortalities. Any abnormalities and mortalities were recorded and the dead fish removed as soon as possible.

Hardness:

113 mg/L CaCO3

Test temperature:

22.9-24.8 degrees Celsius

pH:

7.25-8.10

Dissolved oxygen:

60-100%

Nominal and measured concentrations:

Due to the complexity and low solubility of the mixture, the dissolved test substance was below the limit of detection with the analytical method, therefore the test result was based on a nominal loading rate.

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L/vessel
- Aeration: yes - dechlorination of tap water
- No. of organisms per vessel: 10 fish/test group
- No. of vessels per concentration (replicates): 1 per dose group
- No. of vessels per control (replicates): 1 blank control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water dechlorinated by aeration.

OTHER TEST CONDITIONS
- Adjustment of pH: no (7.25-8.10)
- Photoperiod: 14 hours light / 10 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish were observed for external abnormalities and mortality daily. Any observations were recorded and dead fish were removed as soon as possible.


RANGE-FINDING STUDY
A preliminary test was conducted to determine the test concentrations.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOELR

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Duration:

28 d

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

The pH value was 7.25-8.10; the continuous monitoring temperature was 22.9-24.8 degrees Celsius; the dissolved oxygen was 0-100% respectively.
The concentrations of the test solution could not be quantified and they were below the limit of detecion with this analytical method. At day 4, one fish in the blank control group was found dead making the mortality 10%, which met the criterion for the validity test. Except that, there was no abnormal behaviour or mortality in all the other test groups.
The growth rate of the blank control was 76.5%, and the mean weight had increased by at least 50% of their initial mean weight over 28 days, meeting the validity criteria. The "puesdo" specific growth rate results showed that the 28-day NOERL was 100 mg/L loading rate WAF and the 28-day LOERL was more than 100 mg/L loading rate WAF based on nominal loading rates.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The test did not include replication of tanks at all concentration levels. The estimation of the NOELR/LOELR could be based on the ANOVA on the 'pseudo' specific growth rate for individual fish. The variation of concentration groups were analysed by mean variance, and Dunnett's method was used to estimate NOELR/LOELR of the ratio of growth rate.

Validity criteria fulfilled:

yes

Conclusions:

The 'pseudo' specific growth rate (r3) of 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L and 100 mg/L loading rate WAF were compared with the blank control. The results showed that the 28-day NOELR was 100 mg/L loading rate WAF and the 20-day LOELR was more than 100 mg/L loading rate WAF based on the nominal loading rate.

Executive summary:

This test guideline was desgined to assess the effects of prolonged exposure to chemicals on the growth of juvenile fish.

Juvenile fish in exponential growth phase were placed, after being weighed, in the test chambers and were exposed to a range of sublethal concentrations of the test substance dissolved in water, preferably under semi-static conditions, for 28 days. At the end of the test, the fish were weighed again and the effects on growth rates were analysed using a regression model; the lowest observed effect loading rate (LOELR) and the no observed effect concentration loading rate (NOELR) were determined.

Due to the complexity and low solubility of the mixture, the dissolved test substance was below the limit of detection with the analytical method, therefore the test result was based on a nominal loading rate.  

The 'pseudo' specific growth rate (r3) of 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L and 100 mg/L loading rate WAF were compared with the blank control. The results showed that the 28-day NOELR was 100 mg/L loading rate WAF and the 20-day LOELR was more than 100 mg/L loading rate WAF based on the nominal loading rate.

Reference 2

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2013 to 18 January 2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-700
- Expiration date of the lot/batch: 01 October 2015
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature under nitrogen in the dark

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

A WAF preparation was conducted to give a 102 mg/L (100 mg/L calculated based on 98% purity of the main constituent) loading rate (limit test) and a blank control; these were used in the test directly.

Vehicle:

no

Details on test solutions:

0.51 g of test substance and 500 mL of test water were stirred with a magnetic stirrer for 24 hours to give a nominal concentration of 102 mg/L suspension. After leaving the mixture to stand for 1 hour, the water-accommodated fraction (WAF) was removed by mid-depth siphoning (the first 80-100 mL discarded) to give the 102 mg/L (100 mg/L calculated based on 98% purity) loading rate WAF. A control WAF was also prepared without the test substance.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish
- Strain: Brachydanio rerio
- Length at study initiation (length definition, mean, range and SD): 228.81 +/- 1.52 mm
- Weight at study initiation (mean and range, SD): 0.202 +/- 0.018 g
- Feeding during test: fed with red worm once a day

ACCLIMATION
- Acclimation period: 29 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: fed a minimum of three times a week at least until 24 hours before test initiation
- Health during acclimation (any mortality observed): 0% mortality during 7 days before the test starting date

Test type:

semi-static

Water media type:

other: Dechlorinated tap water

Limit test:

yes

Total exposure duration:

14 d

Remarks on exposure duration:

Evaluating prolonged toxicity over 14 days

Post exposure observation period:

Mortality and visible abnormalities were observated and recorded on days 1, 3, 5, 7, 9, 11, 13 and 14 after the test initiation date. Characterisation, size, behaviour, appearances, dietry levels and changes in body weight and length were recorded.

Hardness:

100 mg/L CaCO3

Test temperature:

23.3-24.7 degrees Celsius

pH:

7.33-8.26

Dissolved oxygen:

64-96 %

Nominal and measured concentrations:

According the results from the Fish Acute Toxicity Test, a WAF of a nominal loading rate of 102 mg/L was used. Due to the low water solubility and complex mixture of EXP0700332, the dissolved test substance was below the detection limit of the analytical method and, therefore, the results were based on nominal loading rates only

Details on test conditions:

TEST SYSTEM
- Test vessel: 4L vessel
- Aeration: No
- No. of organisms per vessel: 10 fish per test group
- No. of vessels per concentration (replicates): 1 vessel per concentration (no replicates) - limit dose
- No. of vessels per control (replicates): 1 vessel per control (no replicates) - 1 blank control
- No. of vessels per vehicle control (replicates): no vehicle control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 hours light / 10 hours dark
- Light intensity: Artificial light of white fluorescent lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality, abnormal responses, body weight and body length

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOELR

Effect conc.:

ca. 102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Duration:

14 d

Dose descriptor:

LOELR

Effect conc.:

> 102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Mortality
The cumulative mortality was 0% for both the blank control and the test group.
- Abnormality observations
No abnormal response was seen for the blank control, nor the nominal loading rate of 102 mg/L WAF test group. LOEL > 102 mg/L and NOEL > 102 mg/L based on the nominal loading rate.
- Body weight and body length
No abnormalities recorded

Results with reference substance (positive control):

not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the 14d threshold level of lethal effect of test substance was greater than 102 mg/L loading rate WAF based on nominal loading rate for Zebra fish (Brachydanio rerio). The 14d Lowest Observed Effect Loading Rate (LOEL) was more than 102 mg/L loading rate WAF based on nominal loading rate. The 14d No Observed Effect Loading Rate (NOEL) was 102 mg/L loading rate WAF based on nominal loading rate.

Executive summary:

The aim of this study was to evaluate the Prolonged Toxicity of EXP0700332 over a 14 -day test period. Zebra fish (Brachydanio rerio) were exposed to aqueous test media containing the test item under defined conditions. Recorded effects were threshold levels of lethality and other observed effects and the NOEL for the test fish.

Due to the low water solubility and complex mixture of EXP0700332, the dissolved test substance was below the detection limit of the analytical method and, therefore, the results were based on nominal loading rates only.

Under the conditions of the test, the 14d threshold level of lethal effect of test substance was greater than 102 mg/L loading rate WAF based on nominal loading rate for Zebra fish (Brachydanio rerio). The 14d Lowest Observed Effect Loading Rate (LOEL) was more than 102 mg/L loading rate WAF based on nominal loading rate. The 14d No Observed Effect Loading Rate (NOEL) was 102 mg/L loading rate WAF based on nominal loading rate.

Description of key information

14 Day Fish Toxicity Study

The study was conducted in accordance with GLP and the OECD 204 guideline. A limit test was performed at 102 mg/L loading rate WAF; the lowest observed effect loading rate (LOELR) was more than 102 mg/L and the no observed effect loading rate (NOELR) was 102 mg/L loading rate WAF based on nominal loading rates.

Juvenile Growth Test

The study was conducted in accordance with GLP and the OECD 215 guideline. One blank control and five test concentrations of 10 mg/L, 18 mg/L, 32 mg/L, 56 mg/L and 100 mg/L loading rate WAFs were prepared. The LOELR was more than 100 mg/L and the NOELR was 100 mg/L loading rate WAF based on nominal loading rates.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

102 mg/L