Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-06 to 2014-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with a minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results. The study plan states that the 'feeding will be discontinued 24 hours prior to testing" for the stock fish to be used in the test. However, the records indicate that the stock fish were fed approx 23- 25.5 hours prior the start of the test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

feeding stopped before time specified in study plan

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

as above

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

Range-finding study
Stability - A sample of the 100 mL loading rate WAF was taken for analysis at 0 and 24 hours. All samples were stored at approximately -20°C prior to analysis.

Definitive test
Concentration and stability - Samples take at 0 and 72 hours (fresh media), and at 24 and 96 hours (old media)
Water samples taken from control and 100 mg/L loading rate WAF test group at 0 and 72 hours (fresh media), and at 24 and 96 hours (old media) for quantitative analysis. All samples were stored at approximately -20°C prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media), were taken and stored at approximately -20°C for further analysis if necessary.

Details on test solutions:

Validation of mixing period
Pre-study investigational work was performed to determine if stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF.
A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for 95 hours. After a 1-hour standing period, the mixtures were removed by siphon and samples taken for TOC.

Range-finder
A nominal amount of test item (2200 mg) was added to the surface of 22 litres of test water to give the 100 mg/L loading rates. After the addition of the test item the 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 aafter 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was justified to remove the WAF by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 - 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations were performed on the WAF after filtering and showed no dispersed test item present.
Definitive test
A limit test at a single loading rate of 100 mg/L was conducted to confirm the range-finder results.
The test concentration was prepared in the same manner as for the range finder.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 21 March 2014. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 02 April 2014 to 14 April 2014. 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 14°C with a dissolved oxygen content of greater than or equal to 9.0 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 23 hours prior to the start of the definitive test. Whilst this was a deviation to the study plan which states that feeding will be discontinued 24 hours prior to the start of testing this was considered not to impact the test as no adverse effects were observed during testing. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.0 cm (sd = 0.1) and a mean weight of 1.05 g (sd = 0.08) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.37 g bodyweight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test Water
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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The test vessels were sealed and maintained at approximately 14ºC.
the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

7.8 - 8.3

Dissolved oxygen:

9.8 - 10.4 mg O2/L

Salinity:

No applicable

Nominal and measured concentrations:

nominal: 100 mg/L loading rate WAF

Details on test conditions:

In the definitive test 25- 30 liter glass exposure vessels containing 20 L test media were used for the test concentration. At the start of the test seven fish were placed in the test vessel. The test vessel was then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.
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.

Reference substance (positive control):

no

Key result

Duration:

96

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: loading rate WAF

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: loading rate WAF

Details on results:

Range-finding test
The results showed no mortalities at the single loading rate of 100 mg/L employed in the test.

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 24 hours showed that measured concentrations of 0.0037 and 0.0031 mg/L were obtained respectively indicating that the test item was stable under test conditions. Due to inconsistencies during validation, the method was amended prior to the definitive test, therefore the range finding samples were analysed using a different method to that of the definitive test, which had a slightly higher LOQ.

Definitive test
Chemical analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0039 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Mortality Data:
There were no mortalities in 7 fish exposed to a test concentration of 100 mg/L loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h) LL50 (mg/L loading rate WAF)

3 >100
6 >100
24 >100
48 >100
72 >100
96 >100

The No Observed Effect Concentration (NOEC) was 100 mg/L loading rate.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in the test.

Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was >=60% of ASV in the control and test vessels.

Results with reference substance (positive control):

Not applicable

Sublethal observations / clinical signs:

Validation of mixing period

Preliminary investigation work in dicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to fish was assessed in accordance with OECD Guideline 203. The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) gave a 96-Hour LL50 of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a water accommodated fraction (WAF)

Following a preliminary range-finding test, seven fish were exposed to aWAF of the test item, at a single nominal concentration of 100 mg/L loading rate WAF for a period of 96 hours at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 24 hours showed that measured concentrations of 0.0037 and 0.0031 mg/L were obtained respectively indicating that the test item was stable under test conditions. Due to inconsistencies during validation, the method was amended prior to the definitive test, therefore the range finding samples were analysed using a different method to that of the definitive test, which had a slightly higher LOQ.

Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave a LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L

Description of key information

The acute toxicity of the test material to fish was assessed in accordance with OECD Guideline 203.  Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave a LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The key study was performed on the reference substance. The study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a water accommodated fraction (WAF)

Following a preliminary range-finding test, seven fish were exposed to aWAF of the test item, at a single nominal concentration of 100 mg/L loading rate WAF for a period of 96 hours at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 24 hours showed that measured concentrations of 0.0037 and 0.0031 mg/L were obtained respectively indicating that the test item was stable under test conditions. Due to inconsistencies during validation, the method was amended prior to the definitive test, therefore the range finding samples were analysed using a different method to that of the definitive test, which had a slightly higher LOQ.

Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave a LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.