Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

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:

15 December 2017 to 22 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and controls. Analytical sampling in a test item loading rate was stopped after the next sampling interval after occurrence of 100% mortality.
- Sampling method: Analytical samples were taken in 24 h intervals from fresh and aged test solutions.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical test site.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Watyer Accommodated Fraction (WAF) - Based on the results of a GLP range-finding test, the following nominal test loading rates were used: control, 2.13, 4.70, 10.3, 22.7 and 50 mg/L. Stock solutions were prepared by directly weighing 75.0, 34.05, 15.45, 7.05 and 3.20 mg in 1500 mL test medium, respectively. These stock solutions were stirred in the dark for 48 h, at room temperature and settled down for 1 h (based on OECD Series on Testing and Assessment No. 23).
- Eluate: The test medium was water composed of reconstituted test water consisting of analytical grade salts dissolved in purified water. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity, with a conductivity of 680 µS/cm.
- Differential loading: 2.13, 4.70, 10.3, 22.7 and 50 mg/L (WAF)
- Controls: Yes, test medium control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After stirring and setlling, a tyndall effect was observed in all test item concentrations, which disappeared in 2.13, 4.70 and 10.3 mg/L after centrifugation (5000 rpm for 1 h). In 22.7 and 50 mg/L solutions, a tyndall effect could still be observed.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Not reported
- Source: Fraunhofer IME (Schmallenberg, Germany)
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): 21.9 mm (21.4 - 22.0 mm; SD = 1.9)
- Weight at study initiation (mean and range, SD): 0.0881 g (0.0724 - 0.1132 g; SD = 0.0278)
- Method of breeding: in house rearing, no further details provided
- Maintenance of the brood fish: The holding water was composed of reconstituted test water consisting of analytical grade salts dissolved in purified water. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The water parameters were controlled at least 2 times a week. The pH-value of the culturing water was within a range of 6.0 – 8.5. The dissolved oxygen was above 80 % of the air saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The fish were cultured at a constant temperature of 21 to 25 °C within a range of 2 °C in a climate controlled room with 16 hours of illumination and 8 hours of darkness.

ACCLIMATION
- Acclimation period: Not reported (fish used were reared in the laboratory)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: The animals were fed with commercial fish flake food (TetraMin) and protein-enriched pellet food (Caviar).
The fish were fed each day with granular rearing food with approx. 2 % of their body-weight. The food was obtained from the fish supplier.
- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed): Only fish in good health and free from any apparent malformation were used. No report of mortality in stock fish

QUARANTINE (wild caught)
- Not applicable

FEEDING DURING TEST
- Not applicable

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Standard guideline exposure duration

Hardness:

13°dH corresponding to 231 mg CaCO3/L

Test temperature:

13°dH corresponding to 231 mg CaCO3/L

pH:

7.33 - 7.73

Dissolved oxygen:

≥ 62 % of air saturation

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal Loading Rates: 2.13, 4.70, 10.3, 22.7 and 50 mg/L, including an untreated control

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Material, size, headspace, fill volume: Glass filled with 1.5 L test solution
- Aeration: continuous aeration of the test tanks with a membrane pump using a Pasteur pipette after 48 h
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 0.53 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium was water composed of reconstituted test water consisting of analytical grade salts dissolved in purified water. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity, with a conductivity of 680 µS/cm.
- Intervals of water quality measurement: The pH-value, temperature and O2 saturation were measured in 24 hour intervals in fresh and aged test solutions. Total hardness was measured at test start.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light, 8h dark
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were observed at 0, 4, 24, 48, 72 and 96 hours after test start. Fish were considered dead if there was no visible movement (e.g. gill movement), and if touching of the caudal peduncle produced no reaction. Records were made on visible abnormalities as: loss of equilibrium, swimming behaviour, respiratory function, pigmentation and all other observed events. Dead fish were removed if observed and mortality, length and weight was recorded. At termination of the test, all remaining fish were euthanized and all fish were weighed and measured.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

15.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No confidence limits obtainable

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: All fish were weighed and measured (see Appendix A, Table 3). The average weight of the test organisms was 0.0881 - 0.0278 g; the average length was 21.9 - 1.9 mm.
- Other biological observations: No sublethal effects were observed in the control and at the test item loading rates up to and including 10.3 mg/L. At 22.7 mg/L one fish showed loss of equilibrium after 4h. Six fish were dead. After 24h, all fish had died. At 50.0 mg/L, three fish showed mild symptoms like reduced activity at test start. Two fish displayed loss of equilibrium. After 4h, all fish were dead.
- Mortality of control: No
- Other adverse effects control: None reported
- Abnormal responses: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The LL50-value after 4 h was calculated by Weibull analysis using linear max. likelihood regression.
The LL50-values after 24, 48, 72 and 96 h were calculated as the geometric mean between the highest test item loading rate causing no mortality and the lowest test item loading rate causing 100 % mortality.
Only loading rates within a clear dose response were used for calculations. For data evaluation the statistical programme ToxRat Professional 3.2.1 was used. The level of significance for all statistical procedures was set to α = 0.05.
The NOELR (mortality) was established based on the highest test loading rate at which no mortality above the allowed control mortality was observed.

Sublethal observations / clinical signs:

Analytical Results

The content of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) in test solutions was analytically determined to confirm the correct application of the test item. The measured content of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) in fresh test solutions was between 87 % and 109 % of nominal. The measured content of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) in aged test solutions was between 83 % and 104 % of nominal. Since the measured concentrations of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) in all test item solutions were between 80 and 120 % of nominal concentrations, the biological endpoints were evaluated using nominal test item concentration.

Table 1. % Mortality of fish in the test

Test Item Loading Rate (mg/L)	Control	2.13	4.70	10.3	22.7	50.0
Time (h)	Mortality (%)	Mortality (%)	Mortality (%)	Mortality (%)	Mortality (%)	Mortality (%)
4	0	0	0	0	86	100
24	0	0	0	0	100	100
48	0	0	0	0	100	100
72	0	0	0	0	100	100
96	0	0	0	0	100	100

Table 2. Determined concentrations of  Mortality (%) Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) during the test

Test Item Nominal (mg/L)	Sampling	Concentration (mg/L)	% Nominal	Mean fresh (%)
0	0 h fresh	< LOD	-	-
0	96 h aged	< LOD	-	-
2.13	0 h fresh	1.85	87	-
2.13	96 h aged	1.76	83	87
4.70	0 h fresh	5.11	109	-
4.70	96 h aged	4.91	104	109
10.3	0 h fresh	10.1	98	-
10.3	96 h aged	10.3	100	98
22.7	0 h fresh	22.8	100	-
22.7	96 h aged	23.6	104	100
50.0	0 h fresh	48.8	98	-
50.0	96 h aged	47.2	94	98

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the LL50 (96 h) of the test item was determined to be 15.3 mg/L test item (nominal). The corresponding NOELR (mortality) (96 h) was 10.3 mg/L test item (nominal).
No sublethal effects were observed in the control and test item loading rate up to and including 10.3 mg/L test item (nominal) after 96 h.

Executive summary:

A study was conducted to determine the effects of the test item to the freshwater zebrafish Danio rerio, conducted in accordance with OECD 203. The test item was evaluated in a static concentration response test, 96 h exposure in treated test medium; 7 organisms per test loading rate were used. Assessments on effects and mortality after 0, 4, 24, 48, 72 and 96 hours were conducted. Following a static GLP range-finding test, the following nominal test item loading rates were tested: Control, 2.13, 4.70, 10.3, 22.7 and 50.0 mg/L. The test item is a complex substance (UVCB) composed of constituents with varying water solubility, therefore the test item is applied as water accommodated fraction (WAF). Temperature, pH-value and % oxygen saturation of the test solutions, measured after 0, 24, 48, 72 and 96 hours from fresh and aged test solutions, are reported. Hardness of the test medium was measured at the start of the test. Analytical samples taken at t = 0 h fresh and t = 96 h aged from control, 2.13, 4.70 and 10.3 mg/L were analysed. Additionally, samples taken at t = 0 h fresh and t = 24 h aged  were analysed from 22.7 and 50.0 mg/L. The NOELR (mortality) was established based on the highest test item loading rate at which fish are alive within the allowed control mortality. The LL50 (mortality) was established as the test item loading rate causing 50 % mortality in the test organism population. The LL50-value after 4 h was calculated by Weibull analysis using linear max. likelihood regression. The LL50-values after 24, 48, 72 and 96 h were calculated as the geometric mean between the highest test item loading rate causing no mortality and the lowest loading rate causing 100 % mortality.

Control mortality: The mortality in the control should not exceed one fish at the end of the test. In this study there was no control mortality. Oxygen saturation: The dissolved oxygen saturation in control and test vessels should be at least 60 % of the air saturation value throughout the test. In this test, the dissolved oxygen concentration was ≥ 62 % throughout the test. The total hardness (as CaCO3) of the test water was determined to be 13°dH; the mean pH-value of the untreated control was determined to be 7.42  0.12. The mean temperature of the untreated control and test item concentrations was measured to be 24.0 +/- 0.2 °C and the oxygen saturation was determined to be 88 +/- 12 %. The content of the active ingredient in test solutions was analytically determined to confirm the correct application of the test item. The measured content of the active ingredient in fresh test solutions was between 87 % and 109 % of nominal. The measured content of the active ingredient in aged test solutions was between 83 % and 104 % of nominal. Since the measured concentrations of the active ingredient in all test item solutions were between 80 and 120 % of nominal concentrations, the biological endpoints were evaluated using nominal test item loading rates. According to the results of the test, the LL50 (96 h) of the test item was determined to be 15.3 mg/L (nominal). The corresponding NOELR (mortality) (96 h) was 10.3 mg/L (nominal). No sublethal effects were observed in the control and test item loading rate up to and including 10.3 mg/L test item (nominal) after 96 h.

Description of key information

96h LL50 = 15.3 mg/L; OECD 203 - Danio rerio; Schiller, 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

15.3 mg/L

Additional information

OECD 203 (2018): A study was conducted to determine the effects of the test item to the freshwater zebrafish Danio rerio, conducted in accordance with OECD 203. The test item was evaluated in a static concentration response test, 96 h exposure in treated test medium; 7 organisms per test loading rate were used. Assessments on effects and mortality after 0, 4, 24, 48, 72 and 96 hours were conducted. Following a static GLP range-finding test, the following nominal test item loading rates were tested: Control, 2.13, 4.70, 10.3, 22.7 and 50.0 mg/L. The test item is a complex substance (UVCB) composed of constituents with varying water solubility, therefore the test item is applied as water accommodated fraction (WAF). Temperature, pH-value and % oxygen saturation of the test solutions, measured after 0, 24, 48, 72 and 96 hours from fresh and aged test solutions, are reported. Hardness of the test medium was measured at the start of the test. Analytical samples taken at t = 0 h fresh and t = 96 h aged from control, 2.13, 4.70 and 10.3 mg/L were analysed. Additionally, samples taken at t = 0 h fresh and t = 24 h aged  were analysed from 22.7 and 50.0 mg/L. The NOELR (mortality) was established based on the highest test item loading rate at which fish are alive within the allowed control mortality. The LL50 (mortality) was established as the test item loading rate causing 50 % mortality in the test organism population. The LL50-value after 4 h was calculated by Weibull analysis using linear max. likelihood regression. The LL50-values after 24, 48, 72 and 96 h were calculated as the geometric mean between the highest test item loading rate causing no mortality and the lowest loading rate causing 100 % mortality.

Control mortality: The mortality in the control should not exceed one fish at the end of the test. In this study there was no control mortality. Oxygen saturation: The dissolved oxygen saturation in control and test vessels should be at least 60 % of the air saturation value throughout the test. In this test, the dissolved oxygen concentration was ≥ 62 % throughout the test. The total hardness (as CaCO3) of the test water was determined to be 13°dH; the mean pH-value of the untreated control was determined to be 7.42 +/- 0.12. The mean temperature of the untreated control and test item concentrations was measured to be 24.0 +/- 0.2 °C and the oxygen saturation was determined to be 88 +/- 12 %. The content of the active ingredient in test solutions was analytically determined to confirm the correct application of the test item. The measured content of the active ingredient in fresh test solutions was between 87 % and 109 % of nominal. The measured content of the active ingredient in aged test solutions was between 83 % and 104 % of nominal. Since the measured concentrations of the active ingredient in all test item solutions were between 80 and 120 % of nominal concentrations, the biological endpoints were evaluated using nominal test item loading rates. According to the results of the test, the LL50 (96 h) of the test item was determined to be 15.3 mg/L (nominal). The corresponding NOELR (mortality) (96 h) was 10.3 mg/L (nominal). No sublethal effects were observed in the control and test item loading rate up to and including 10.3 mg/L test item (nominal) after 96 h.