Hexadecyltrimethylammonium methyl sulphate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 27 August, 2012 to 31 August, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Chemical analyses
Samples were taken for chemical analysis from the stock and all test concentrations at the start and end of the test as well as in the old and new test solutions after 48 h.
Samples of 10 mL were taken and diluted 1:1 in leaching solution. Leaching solution used was 100 g/L of MgCl2.6H20 dissolved in a 50% methanol and 50%
2-propanol solution. Samples were shaken for a short period on a shaking table and further diluted (also in leaching solution) if required to fall within the calibration curve. Chemical analysis was conducted according to the procedure.

Vehicle:

no

Details on test solutions:

Preparation of the stock solution
1.5027 g of the test substance was weighed out using the analytical balance for preparation of the stock solution. 300 mL of the test medium was added and the solution was stirred at room temperature. The test chemical dissolved easily and formed a clear and homogeneous stock solution. The stock solution was then made up to 2.5 litres and the pH was adjusted slightly with HCl and the stock was further filled to 3 litres. The stock solution was then used to generate the chosen test concentrations and was kept stirring when in use.

Preparation of the test solutions
An appropriate amount of the stock solution required to reach the desired concentration in a total volume of 3 L was pipetted into each 3 L aquarium. The aquarium was then brought up to exactly 3 L volume with a volumetric flask and homogenized with a Teflon coated magnetic stirrer. The following test concentrations were prepared:

0, 10, 22, 48.4, 106.5 and 234.3 mg/L test substance

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Strain: Danio rerio
- Source: “Dierenvriend” in Arnhem, The Netherlands
- Food type: The fish in stock were fed one to three times per day with commercially available dry, deep frozen food, or fresh Artemia salina nauplii /juvenile Daphnia magna neonates. Feeding was stopped 24 hours before the test was started.
- Length at study initiation: >3 cm
- Weight at study initiation (mean and range, SD): 0.3 g
- Feeding during test: No

ACCLIMATION
- A representative number of test fish batch (10 at random) were weighed prior to the test and measured after the test to assess compliance with guideline criteria.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Dutch Standard Water: 12.3 ºdH (the guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH)

Test temperature:

Temperature controlled incubator: 21.3-22°C

pH:

8.2

Dissolved oxygen:

>8.4 mg/L

Salinity:

Dutch Standard Water: 200 mg of CaCl2.2H2O, 180 mg of MgSO4.7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3.

Nominal and measured concentrations:

Nominal: 0, 10, 22, 48.4, 106.5 and 234.3 mg/L.

Details on test conditions:

Test vessel:
3-L aquaria (capacity slightly exceeded 3 L) containing 3 L of test solution were used. They were covered with glass plates and aerated gently during the test.

Test room and light regime
The test was carried out in a temperature controlled room. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.

The dissolved oxygen concentrations and conductivity were determined electrochemically using an oxygen electrode / conductivity electrode and meter. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. Hardness, was measured using a Dr Lange test kit validated against an appropriate standard. TOC measurements were conducted with a Shimatzu TOC apparatus according to laboratory standard operating procedures

Test principle and procedure
The test was performed as a semi static test which means that the test media was replaced after 48 hours to ensure that a worst case scenario was tested. The minimum requirement of 7 fish per test concentration and control were used for ethical reasons.

Under otherwise identical test conditions, the fish were exposed to the chosen concentrations of the test substance and mortality and sub-lethal effects were recorded at least at 30 minutes and approximately 1, 2 and 4 hours after exposure and then and then at approximately 24, 48, 72 and 96 hours. Additional observations after the solution refreshment were made for animal welfare reasons. This data was not used for endpoint determinations.

The fish were considered dead when a lack of opercular movement was observed and touching of the caudal peduncle produced no reaction. Dead fish were removed from the test vessels directly after being observed, which was checked several times up to 4 h on the first day for ethical reasons and at least twice each day on subsequent days. In addition to death, sub-lethal effects such as erratic swimming, loss of reflex, increased excitability, lethargy, changes in physiology, discoloration, pigmentation, excessive mucous production, hyperventilation, opaque eyes, curved spine or hemorrhaging were recorded if observed. Fish that were convulsing or showing other severe forms of distress not considered transient in nature and likely to become more severe before the exposure is terminated, were sacrificed for humane reasons. These fish were treated as having died in the test.

The fish were randomly placed in the test vessels which were positioned in the test room in a random manner. During the test the vessels were covered with glass plates. The test solutions present in the test vessels were aerated with water-saturated air purified by an active coal filter and a cotton filter during testing.

Evaluation of data
The EC50 was calculated with the computer program Toxcalc™ version 5.0.23 using the trimmed Spearman-Karber method for LC 50 determination and the Fishers exact test for NOEC determination.

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 13.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: calculated using the Spearman Karber Graphical Method

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: No lethal effects observed

Reported statistics and error estimates:

The LC50 calculated using the Spearman Karber Graphical Method was calculated as 0.1 mg/L (Time weighted mean corrected for both components).

Sublethal observations / clinical signs:

Results

Preliminary Test

The preliminary study determined the NOEC as 10 mg/L this was sufficient to indicate the test concentration range required for the definitive study.

Toxicity

The results of the test on survival and sub-lethal effects were summarized. All effects are based on the nominal test concentrations. Endpoints are based on 96 hours exposure. Toxcalc 5.0.23 output was reported. The NOEC for the test substance was determined as 10.0 mg/L. One mortality did occur at 10 mg/L in the last hours of the test. This was however not detected as significant with the statistical test used. The LC50 calculated using the Spearman Karber Graphical Method was calculated as 13.9 mg/L.

Prior to death affected animals displayed reduced activity and a hanging tail in comparison to the control. All test concentrations displayed reduced activity in comparison to the control.

The average length of a sample of 7 fish randomly taken from the test animals at the end of the test was 3.4 cm. The mean biomass loading was 0.7 g/L. The results of the length and mass determination are presented in table 2 of the attached report. The general criterion set for the test animals in the study plan and guideline for biomass loading was met. The criterion for length was exceeded slightly.

Survival and sub-lethal effects of fish

Date

27/8

27/8

27/8

27/8

28/8

28/8

28/8

29/8

29/8

29/8

29/8

30/8

31/8

31/8

Time

13:20

13:30

13:50

14:55

17:05

08:10

13:20

16:00

09:30

13:20

16:00

16:00

11:00

13:20

Hours Test

0

24

48

72 (Approx)

96

Control

7

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

7AN

10 mg/L

7

7AN

7AN

7AN

7AN

7RA

7RA

7RA

7RA

7RA

7RA

7RA

7RA

6RA

22 mg/L

7

7AN

5RA 2AN

7RA

1AN 6RA

0

48.4 mg/L

7

7RA

7RA

5D 2#

106.5 mg/L

7

7RA

7#

234.3 mg/L

7

3D 4#

AN=Appearing normal, #=Euthanized for Ethical reasons D=Dead RA=Reduced activity  A=Surfacing

 

Chemical analyses

All recovery was >80% of the nominal for both components and therefore nominal concentrations only were used for the endpoint calculation and the test substance should be considered stable. All quality criteria were met. Analytical parallel replicates were not required and data generated was not used further.

Oxygen, pH, Temperature, Conductivity and Hardness

The oxygen concentrations varied during the test from 7.5 to 8.5 mg/L. The maximum pH variation during the test was from 7.7 to 8.3. The temperature in the test room during the test period ranged from 21.3 to 22.0°C. The conductivity at the start of the test in dilution water was 601 µs/cm. The hardness of the test water was measured as 12.3ºdH which is equivalent of a 216 mg/L as calcium carbonate of the attached report.

The following quality criteria have been met in the present study:

- Mortality in the control did not exceed one fish at the end of the test.

- The dissolved oxygen concentration was >60% of the air saturation value throughout the test.

- Test substance recovery (for both components) was >80% for the entire test duration.

- pH variation did not exceed 1.5 units

All measurements fell within study plan and guideline criteria.

Deviations from the study plan

- The control vessel was analyzed once only not also before and after refreshment and at the end of the test as detailed in the study plan. This is not a critical deviation as was not a test concentration containing test substance and the validity of the results are not affected.

- The average length of the fish used for testing exceeded 3 cm slightly. Due to the biomass loading still comfortably meeting the set criterion this is not expected to significantly affect the results.

- Test solutions were not agitated before sampling as stated in the study plan. Analytical results were however all >80% this is not considered to have affected the results in any way.

Conclusion

Endpoints were calculated or determined as far as possible using the guideline recommended procedures. An LC50 of 13.9 mg/L was calculated and a NOEC of 10.0 mg/L were determined. Analytical measurement of the new solutions indicated that the test material was accurately dosed to the test system and stable throughout the test period. Amendments and Deviations were reported and were not of significant influence to the test outcome. The study can be considered an accurate representation of the effects of the test chemical to D.Rerio in guideline compliant synthetic water.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 96 h LC50 and NOEC were calculated to be 13.9 mg a.i./L (nominal) and 10 mg a.i./L (nominal) respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, C12-14 ADMAES (96% active) to Danio rerio(zebra fish) under semi-static conditions according to OECD 203 Guideline, in compliance with GLP. The read across substance was tested at the nominal concentrations of 0, 10, 22, 48.4, 106.5 and 234.3 mg/L. The concentration of the read across substance in aqueous samples, was analysed at the beginning, middle and end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). Chemical analysis from the stock, and all test concentrations at the start and end of the test as well as in the old and new test solutions, were performed after 48 h. All recoveries were above 90% and nominal concentrations were therefore used for endpoint determination. The general validity criterion of the study was met. Under the study conditions, the 96 h LC50 and NOEC were calculated to be 13.8 mg a.i. /L (nominal) and 10 mg a.i./L (nominal) respectively (Kean, 2012). Based on the results of the read across study, similar effect levels can be considered for the test substance.

Description of key information

 Based on the available results of the read across study, the 96 h LC50 of 13.8 mg a.i./L (nominal) for toxicity to freshwater fish has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

13.8 mg/L

Additional information

A study was conducted to determine the acute toxicity of the read across substance, C12-14 ADMAES (96% active) to Danio rerio(zebra fish) under semi-static conditions according to OECD 203 Guideline, in compliance with GLP. The read across substance was tested at the nominal concentrations of 0, 10, 22, 48.4, 106.5 and 234.3 mg/L. The concentration of the read across substance in aqueous samples, was analysed at the beginning, middle and end of the test. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS/MS). Chemical analysis from the stock, and all test concentrations at the start and end of the test as well as in the old and new test solutions, were performed after 48 h. All recoveries were above 90% and nominal concentrations were therefore used for endpoint determination. The general validity criterion of the study was met. Under the study conditions, the 96 h LC50 and NOEC were calculated to be 13.8 mg a.i. /L (nominal) and 10 mg a.i./L (nominal) respectively (Kean, 2012). Based on the results of the read across study, similar effect levels can be considered for the test substance.