bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

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:

24 December 2014 to 15 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

TEST WATER
- The test water used for the definitive test 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 passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

VALIDATION OF THE MIXING PERIOD
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF (see Appendix 3, attached).

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, Hebden, near Skipton, Yorkshire, UK and maintained in-house from 23 March 2015.
- Fish were maintained in a glass fibre tank with a 'single pass' water renewal system.
- Fish were acclimatised to test conditions from 20 April 2015 to 27 April 2015.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with a 20 minute dawn and dusk transition period.
- The stock fish were fed commercial trout pellets, which were discontinued approximately 24 hours prior to the start of the definitive test.
- There were 5 mortalities in the 7 days prior to the start of the test and the fish had a mean standard length of 4.5 cm (sd = 0.3) and a mean weight of 1.24 g (sd 0.21) at the end of the definitive test. Based on the mean weight value, the loading rate was 0.43 g bodyweight/L.
- 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

Post exposure observation period:

Not applicable

Hardness:

140 mg/L as CaCO3

Test temperature:

15 °C

pH:

7.5 to 8.3

Dissolved oxygen:

9.0 to 10.3 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

1.0 mg/L (nominal loading rate)

Details on test conditions:

RANGE FINDING TEST
- The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
- A nominal amount of test item (2200 mg) was added to the surface of 22 L of test water to give the 100 mg/L loading rate.
- After addition of test item, the test water was stirred by magnetic stirrer using a stirring rate such that the vortex was formed to give a dimple at the water surface.
- 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.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- 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.
- During discussion with the sponsor prior to testing, it was agreed to test at a loading rate of 1.0 mg/L, however the range-finding test was conducted at a loading rate of 100 mg/L in error. Given that no effect was observed, this still allowed for determination of the loading rate to be employed in the definitive test, therefore this had no impact on the outcome of the study.
- In the range-finding test, three fish were placed in each test and control vessel and maintained in a temperature controlled room at 15 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 96 hours under static test conditions.
- Each 25-30 L test and control vessel contained 20 L of test media and was covered to reduce evaporation.
- After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspction of the test fish.
- The control group was maintained under identical condition but not exposed to the test item.

DEFINITIVE TEST
- A study had been previously conducted in which it was determined that the water solubility of the test item was less than 5.59 x 10E-05 g/L at 20.0 ± 0.5 °C, suggesting that lading rates of greater than 1.0 mg/L were far in excess of that required to ensure the maximum dissolved test item concentration was obtained. Given this, at the request of the sponsor, the definitive test was conducted at a maximum loading rate of 1.0 mg/L.
- A nominal amount of test item (22 mg) was added to the surface of 22 L of test water to give the 1.0 mg/L loading rate.
- After addition of test item, the test water was stirred by magnetic stirrer using a stirring rate such that the vortex was formed to give a dimple at the water surface.
- 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.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF.
- As in the range-finding test, 25-30 L glass exposure vessels containing 20 L of test media were used for the control and 1.0 mg/L loading rate WAF test concentration.
- At the start of the test seven fish were placed in each test vessel containing test preparation at random and maintained at approximately 15 °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 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 which daily renewal of the test preparations took place to prevent build up of nitrogenous waste products.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Appendix 3) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-hour settlement period.

RANGE-FINDING TEST
- Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 (attached).
- The results showed no mortalities at the single loading rate of 100 mg/L WAF.
- Chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed that a measured concentration of 0.0928 mg/L was obtained and at 24 hours showed that a measured concentration of 0.110 mg/L (see Appendix 4, attached).

CHEMICAL ANALYSIS OF TEST LOADING RATES IN THE DEFINITIVE TEST
- Chemical analysis of the freshly prepared 1.0 mg/L loading rate test preparation at 0 and 72 hours showed that measured concentrations of 0.143 and 0.0923 mg/L were obtained respectively. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of 0.0774 and 0.0714 mg/L were obtained respectively (see Appendix 4, attached).

MORTALITY DATA
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 (below).
- There were no mortalities in 7 fish exposed to a 1.0 mg/L loading rate WAF for a period of 96 hours.
- Inspection of mortality data gave LL50 values > 1.0 mg/L loading rate WAF at 3, 6, 24, 48, 72 and 96 hours.
- The No Observed Effect Loading Rate (NOELR) was considered to be 1.0 mg/L loading rate WAF.

SUB-LETHAL EFFECTS
- There were no sub-lethal effects of exposure observed in 7 fish exposed to a 1.0 mg/L loading rate WAF for a period of 96 hours.

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.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at approximately 15 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start of each mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAF.
- At the start of the mixing period the 1.0 mg/L loading rate was observed to be a clear colourless water column with globules of test item floating at the surface.
- After 23 hours stirring and a 1 hour standing period, the 1.0 mg/L loading rate was observed to remain as at the start of stirring.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, the 1.0 mg/L loading rate was observed to be a clear, colourless, solution with the exception of the fresh media at 0 hours and the corresponding old media at 24 hours, which was observed to be a clear colourless solution.

Sublethal observations / clinical signs:

Table 2: Cumulative mortality data in the definitive test
Nominal Loading Rate (mg/L)	Cumulative mortality (initial population = 7)						% Mortality
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours	96 hours
Control	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. The study showed that the test item was not toxic to rainbow trout at a concentration in excess of the water solubility of the substance.

Executive summary:

GUIDELINE

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

METHOD

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 1.0 mg/L for a period of 96 hours at a temperature of approximately 15 °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 freshly prepared 1.0 mg/L loading rate test preparation at 0 and 72 hours showed that measured concentrations of 0.143 and 0.0923 mg/L were obtained respectively. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of 0.0774 and 0.0714 mg/L were obtained respectively.

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

CONCLUSION

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. The study showed that the test item was not toxic to rainbow trout at a concentration in excess of the water solubility of the substance.

Description of key information

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. The study showed that the test item was not toxic to rainbow trout at a concentration in excess of the water solubility of the substance (OECD 203 and EU Method C.1).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1 mg/L

Additional information

GUIDELINE

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

METHOD

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 1.0 mg/L for a period of 96 hours at a temperature of approximately 15°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 freshly prepared 1.0 mg/L loading rate test preparation at 0 and 72 hours showed that measured concentrations of 0.143 and 0.0923 mg/L were obtained respectively. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of 0.0774 and 0.0714 mg/L were obtained respectively.

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

CONCLUSION

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. The study showed that the test item was not toxic to rainbow trout at a concentration in excess of the water solubility of the substance.