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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2013 to 17 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity 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: 01 October 2015
- Purity test date: 98%

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

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

The test item was stored at ambient temperature in the dark before conducting the study. A nominal concentration of 102 mg/L of test substance was prepared by adding 5000 mL of test water to 0.51 g of test item in a 5 L vessel. The suspension was stirred for 24 hours with a magnetic stirrer at a stirring rate such that an approximately 1 cm depth vortex was formed at the water surface. After 24 hours the stirring was stopped and the mixture was allowed to stand for 1 hour. The WAF (Water-accommodated Fraction) 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 of the main constituent) loading rate WAF and was used in the test directly. 10 fish were exposed for 96 hours with no replicates; additionally, 10 fish were exposed to a blank control with no test substance at the same time. Mortality and visible abnormalities were recorded at 24 hours, 48, hours, 72 hours and 96 hours after being dosed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

A water-accommodated fraction (WAF) was prepared by adding 5000 mL of test water to 0.51 g of test item in a 5 L vessel. The suspension was stirred for 24 hours with a magnetic stirrer at a stirring rate such that an approximately 1 cm depth vortex was formed at the water surface. After 24 hours the stirring was stopped and the mixture was allowed to stand for 1 hour. The WAF (Water-accommodated Fraction) 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 of the main constituent) loading rate WAF and was used in the test directly. A control WAF was also prepared wihout the test item.

Test organisms (species):

other: Brachydanio rerio

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Guangahou Yangshi Breeding Farm
- Weight: 0.199 - 0.279 g
- Length: 23.96 - 25.46 mm

ACCLIMATION
- Acclimation period: 22 days
- Acclimation conditions: Temperature: 23 +/- 2 degrees Celsius; 14 hours light / 10 hours dark; Dissolved oxygen concentration at least 80% or air saturation value
- Feeding frequency during acclimation: Fed daily until 24 hours before test initiation
- Health during acclimation (any mortality observed): No mortality was observed 7 days before test initiation.

Test type:

semi-static

Water media type:

other: Dechlorinated tap water

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Mortality and visible abnormality of the test fish were recorded at 24 hours, 48 hours, 72 hours and 96 hours after the start of the test period. Dead fish (no visible movement and if touching of the caudal peduncle produced no reaction) were removed as soon as they were found.

Hardness:

100 mg/L CaCO3

Test temperature:

23.8 - 24.7 degrees Celsius

pH:

7.41 - 8.01

Dissolved oxygen:

At least 60% of the air saturation

Nominal and measured concentrations:

The dissolved test substance was below the detection limit of the analytical method, therefore the results were based on nominal loading rates only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 4 L/ test vessel
- Aeration: No
- No. of organisms per vessel: 10 fish / test group
- No. of vessels per concentration (replicates): 1 vessel of 102 mg/L nominal loading rate - no replicates
- No. of vessels per control (replicates): 1 control vessel - no replicates
- No. of vessels per vehicle control (replicates): none - no vehicle control
- Biomass loading rate: loading of test fish: 0.585 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Intervals of water quality measurement: The temperature, pH and dissolved oxygen of each test group were measured at 0h, 24h, 48h, 72h and 96h (before and after the renewal) after test initiation.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Artificial light of white fluorescent lamp, 14h light / 10h darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- observation of test organism:
mortality and visible abnormalities recorded at 24h, 48h, 72h and 96h after test initiation.
- temperature, pH and dissolved oxygen
The temperature, pH and dissolved oxygen of each group were measured at 0h, 24h, 48h, 72h and 96h, before and after the renewal, after the test started.
- body weight and length of fish test
the body weight and length of the blank control were measured at the end of the test.
- measurement of actual concentrations of the test substance in test solution
The nominal loading rate of 102 mg/L WAF test group and blank control were measured at 0h, 24h, 48h, 72h and 96h (before and after the renewal) after test initiation by HPLC.

TEST CONCENTRATIONS
The range-finding study showed that there were no mortalities in blank control and the nominal concentration of a 100 mg/L WAF loading rate for a period of 96 hours. Based on this result and the water solubility test, the limit test was conducted with a WAF nominal loading rate of 102 mg/L (taking into account the purity of the test item (98%)).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

> 102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

> 102 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The cumulative mortality of each group at 24h, 48h, 72h and 96h after dose initiation was 0% for both the control group and the limit dose. No abnormal responses were observed during any of the time points for both the blank control and the test group. The body weight and length of the test fish in the blank control group had an average of 0.234 g +/- 0.020 g and 24.91 mm +/- 0.40 mm respectively. The temperature, pH and dissolved oxygen ranged from 23.8 - 24.7 degrees Celsius, 7.41 - 8.01 and 62-98% respectively.

Results with reference substance (positive control):

Not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the 96h-LL50 of test substance to Zebra fish (Brachydanio rerio) was greater than 102 mg/L loading rate WAF based on the nominal loading rate. The highest concentration causing 0% mortality at 96 hours (LL0) was 102 mg/L loading rate WAF based on the nominal loading rate, lowest concentration causing 100% mortality at 96 hours (LL100) was greater than 102 mg/L loading rate WAF based on nominal loading rate.

Executive summary:

The aim of this study is the evaluation of the acute toxicity of the test substance (EXP0700332) to fish ovr a 96 -hour test period. Zebra fish (Brachydanio rerio) were exposed to aqueous test media containing the test item at a 0 and 100 mg/L under defined conditions. Recorded effects were the mortality and visible abnormalities of the test fish.

Due to the complex mixture and poor solubilty of EXP077332, the dissolved test substance was below the detection limit of the analytical method, therefore the results were based on nominal loading rates only.

Under the conditions of the test, the 96h-LL50 of test substance to Zebra fish (Brachydanio rerio) was greater than 102 mg/L loading rate WAF based on the nominal loading rate. The highest concentration causing 0% mortality at 96 hours (LL0) was 102 mg/L loading rate WAF based on the nominal loading rate, lowest concentration causing 100% mortality at 96 hours (LL100) was greater than 102 mg/L loading rate WAF based on nominal loading rate.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2009 and 11 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Modern GLP study conducted in accordance with OECD test guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
no data

Details on sampling:

Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and
the concentration of the test material in the 100 mg/l loading rate WAFs was verified by chemical analysis.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF). An amount of test material (2100 mg) was added to the surface of 21 litres of dechlorinated tap water to give the 100 mg/l loading rate. After the addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present. This method of preparation was conducted in duplicate to give replicates R1 and R2. The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited and maintained in-house since 30 July 2009. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 26 August 2009 to 7 September 2009. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with
20 minute dawn and dusk transition periods. The water temperature was controlled at approximately 14C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.3 cm (sd = 0.3) and a mean weight of 1.81 g (sd = 0.30) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.63 g bodyweight/litre. The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

approximately 140 mg/l as CaCO3

Test temperature:

14o C to 16o C

pH:

7.4 – 7.9

Dissolved oxygen:

8.7 to 9.8 mg O2/l

Salinity:

no data

Nominal and measured concentrations:

100 mg/l loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 20L
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2

TEST MEDIUM / WATER PARAMETERS
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish. Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener)
giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (see attached).

OTHER TEST CONDITIONS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and
the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI93510 digital thermometer.


TEST CONCENTRATIONS

- Range finding study: A range-finding test conducted using a solvent spike method of preparation at a single concentration of 0.70 mg/l showed no adverse effects of exposure. Subsequent to this range-finding test, additional information provided by the Sponsor confirmed that the test material was a complex mixture and hence the use of a solvent spike method of preparation was not applicable. A range-finding test conducted for the Acute Toxicity to Daphnia magna study using a WAF method of preparation showed no toxicity at a loading rate of 100 mg/l.
A range-finding test conducted for the Acute Toxicity to Daphnia magna study using a WAF method of preparation showed no toxicity at a loading rate of 100 mg/l. It was therefore considered that toxicity would not be expected at this concentration in the Acute Toxicity to Rainbow Trout study. Therefore, in the interest of animal welfare, it was considered that the conduct of a range-finding test using a WAF preparation was not necessary. Based on the above a Limit test was conducted at a single loading rate of 100 mg/l.

Duration:

3 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Duration:

6 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mortality (fish)

Details on results:

Mortality data- There were no mortalities in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours. The results of the definitive test showed the highest loading rate resulting in 0% mortality to be greater than or equal to 100 mg/l loading rate WAF, the lowest loading rate resulting in 100% mortality to be greater than 100 mg/l loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate.

Sub-lethal effects: There were no sub-lethal effects of exposure observed in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL50* value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

This study was performed to assess the acute toxicity of the test material to rainbow trout (oncorhynchus mykiss). Fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of 14 to 16ºC under semi-static test conditions. The 96-Hour LL50* based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Description of key information

A key GLP compliant study reporting 96h LL50> 100 mg/l loading rate WAF (OECD 203) .

Another study was conducted in 2013 to GLP and compliant with the OECD 203 guideline. Results showed that under the conditions of the test, the 96h-LL50 of test substance to Zebra fish (Brachydanio rerio) was greater than 102 mg/L loading rate WAF based on the nominal loading rate. The highest concentration causing 0% mortality at 96 hours (LL0) was 102 mg/L loading rate WAF based on the nominal loading rate, lowest concentration causing 100% mortality at 96 hours (LL100) was greater than 102 mg/L loading rate WAF based on nominal loading rate.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Two key GLP complaint studies conducted in accordance with the OECD 203 guideline reported that the 96-Hour LL50* based on nominal loading rates was greater than 100 mg/L and 102 mg/L loading rate WAF.