3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.14 (Fish Juvenile Growth Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test the test material was dissolved directly in deionised reverse osmosis water.
Amounts of test material (512 and 1 600mg) were each separately dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 100 ml to give the 512 and 1600 mg/100 ml aqueous stock solutions. Dilutions were prepared from these to give further aqueous stock solutions of 16, 51.2 and 160 mg/ 100 ml. These stock solutions were freshly prepared each day prior to dosing by the dynamic, continuous flow apparatus to give the 0.010, 0.032, 0.10, 0.32 and 1.0 mg/l test concentrations.
Each of the stock solutions was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis on Days 0, 2, 6, 8, 10, 13, 15, 17, 20, 22, 24, 27 and 28. Analysis of the 24-Hour old stock solutions used to prepare the test series was also carried out on Days 0, 2, 6, 8, 10, 13, 15, 17, 20, 22, 24, 27 and 28.

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 inhouse since 20 September 2002. Fish were maintained in a glass tank with a “single pass” water renewal system. Fish were acclimatised to test conditions from 31 October 2002 to 12 November 2002. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Test type:

flow-through

Total exposure duration:

28 d

Hardness:

approx. 120 mg CaCO3/L

Test temperature:

14°C

Dissolved oxygen:

>9.2 mg O2/L

Nominal and measured concentrations:

- Acute range-finding test
1 mg/L (nominal)
- Definitive test
0.010, 0.032, 0.10, 0.32 and 1 mg/L

Details on test conditions:

The water temperature was controlled at 14.0°C with a dissolved oxygen content of greater than or equal to 9.2 mg 02/1. These parameters were recorded daily. The stock fish were fed commercial trout pellets at a minimum ration of 2% bodyweight /day. There were zero mortalities in the 7 days prior to the start of the test. Pre-exposure measurements showed the fish had a mean standard length of 4.8 cm (sd 0.1) and a mean weight of 1.74 g (sd = 0.2). Based on the mean weight value this gave a loading rate at initiation of the study of 0.70 g bodyweight /litre (static volume), 0.10 g of bodyweight/litre (volume in 24 hours) and 7.2 tank volumes per day. The fish were flot individually identified.
The individual fish weights were calculated to be ± 25% of the arithmetic mean weight with the exception of one fish. This one fish in excess of 25% of the mean was considered not to affect the overall results of the test and overall it was considered that the validation criterion for the fish weight had been sufficiently satisfied.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

In the definitive test 40 litre glass exposure vessels were used for each control and test concentration. At the start of the study 16 fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14.0 ± 1.0°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 28 days. The test vessels received no auxiliary aeration, the diluent water supply only was aerated.
The control group was maintained under identical conditions but not exposed to the test material.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

NOEC

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in 1.6%, 8.0%, 6.0%, 7.8% and 17 % inhibition of the tank-average specific growth rate at the test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L respectively over the test period of 0 to 28 days when compared to the control (zero dose) test group.
The 0 to 28-Day EC20 (inhibition of tank-average specific growth rate) was calculated to be 1.0 mg/L.
The Lowest Observed Effect Concentration was 1.0 mg/L.
The No Observed Effect Concentration was 0.32 mg/L.
Based on the mean measured test concentrations over the test period of 0 to 28 days the EC20 (inhibition of tank-average specific growth rate) was estimated to be 0.72 mg/L, the LOEC was 0.72 mg/L and the NOEC was 0.25 mg/L.

Executive summary:

Introduction: A study was performed to assess the effect of the test material on growth of juvenile rainbow trout (Oncorhynchus mykiss).

The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (2000) No 215, “Fish, Juvenile Growth Test” referenced as Method C.14 of Commission Directive 200 1/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods: Following information provided by the Sponsor and a preliminary acute range-finding test, fish were exposed, in groups of 16 to an aqueous solution of the test material over a range of concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L for a period of 28 days at a temperature of 14.0±1.0°C under dynamic test conditions. Any mortalities or adverse reactions to exposure were recorded daily throughout the exposure period. The fish were fed daily at a rate of 4% bodyweight and the length and weight of each fish determined at 0 and 28 days.

Results: Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in 1.6%, 8.0%, 6.0%, 7.8% and 17% inhibition of the tank-average specific growth rate at the nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L respectively over the test period of 0 to 28 days when compared to the control group. Given that 17% inhibition of tank-average specific growth rate was observed at the highest test concentration of 1.0 mg/L an EC20 value could not be calculated. However, based on this information the EC20 value can be estimated to be approximately 1.0 mg/L based on nominal test concentrations. The Lowest Observed Effect Concentration (LOEC) based on nominal test concentrations was considered to be 1.0 mg/l on the basis that at this test concentration significant inhibition of tank-average specific growth rate and significant differences in terms of the “pseudo” specific growth rate were observed when compared to the control group. The No Observed Effect Concentration (NOEC) based on nominal test concentrations was considered to be 0.32 mg/l on the basis that at this test concentration zero mortalities, insignificant inhibition of tank-average specific growth rate, the absence of any sub-lethal effects of exposure and no significant differences in terms of the ‘pseudo” specific growth rate were observed when compared to the control group.

Analysis of the test preparations was performed using a solid phase cartridge extraction procedure followed by high performance liquid chromatography (HPLC). This method was developed as the method used for the Algal Inhibition Test (Safepharm Laboratories Project No. 1190/061), dilution of samples prior to HPLC, was not sensitive enough to quantify the lower concentrations employed in this test.

The recovery analysis performed showed the recoveries to be low at the lower concentrations employed in the test. The low recoveries were considered to be due to incomplete adsorption onto the SPE matrix from the media during the extraction procedure; this effect was significant for the lower test concentrations. Therefore on each sampling occasion procedural recoveries were performed alongside analysis of the test samples. All test sample results were corrected for the procedural recovery rate. The recovery analysis performed on the aqueous stock solutions, used to dose the test system using dynamic flow test conditions, gave results of near nominal of fortification.

The stability analysis performed showed the test material to be stable in test medium and in deionised reverse osmosis water used for the concentrated aqueous stock solutions.

Analysis of the aqueous stock solutions showed that amongst 65 analyses, 63 ranged from 84% to 107% of nominal, 2 were considered as aberrant (195% and 131%).

Analysis of the test preparations throughout the study showed the measured test concentrations to range from less than the limit of quantitation (which was determined to be 0.0042 mg/l) to 121% of nominal value. Overall there was a trend for the measured test concentrations to be lower than 80% of nominal values despite correction for procedural recoveries. These low values were considered to be due to an artefact of the method of analysis, as observed in the recovery analysis, given that the diluent flow rates varied by only ± 4% and the stock solution usage rates varied by ± 6% throughout the duration of the test.

Given this decline in measured test concentrations it was considered justifiable to base the results on the mean measured test concentrations in order to give a “worst case” analysis of the data.

The EC20 (inhibition of tank-average specific growth rate) based on mean measured test concentrations was estimated to be approximately 0.72 mg/1 given that 17% inhibition of tank-average specific growth rate was observed at this concentration.

The Lowest Observed Effect Concentration (LOEC) based on the mean measured test concentrations was 0.72 mg/L as this test group showed significant inhibition of tank-average specific growth rate and significant differences in terms ofthe “pseudo” specific growth rate when compared to the control group

The No Observed Effect Concentration (NOEC) based on the mean measured test concentrations was 0.25 mg/L as this test group showed zero mortalities, insignificant inhibition of tank-average specific growth rate, no sub-lethal effects of exposure and no significant differences (P>0.05) in terms of the “pseudo” specific growth rate when compared to the control group.

Description of key information

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in 1.6%, 8.0%, 6.0%, 7.8% and 17 % inhibition of the tank-average specific growth rate at the test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L respectively over the test period of 0 to 28 days when compared to the control (zero dose) test group.
The 0 to 28-Day EC20 (inhibition of tank-average specific growth rate) was calculated to be 1.0 mg/L.
The Lowest Observed Effect Concentration was 1.0 mg/L.
The No Observed Effect Concentration was 0.32 mg/L.
Based on the mean measured test concentrations over the test period of 0 to 28 days the EC20 (inhibition of tank-average specific growth rate) was estimated to be 0.72 mg/L, the LOEC was 0.72 mg/L and the NOEC was 0.25 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.25 mg/L