N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2014 to 16 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the determination of the test item concentrations, duplicate samples were taken from each treatment at the start and end of each test medium renewal period. However, from the dilution 1:4.6, the last samples were taken after 24 hours, since all daphnia were immobile at this observation date at this concentration. For the 24- and 48-hour samples, the contents of the respective replicates were combined prior to sampling.
Immediately after sampling, acetonitrile (10 mL acetonitrile per 10 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

The concentrations of the test item were determined in one test medium sample per test concentration and control from all sampling dates or from the sampling time at the end of the respective exposure period when all daphnia were immobile.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (with respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.

At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in August 2014 (48-hour EC50: 1.0 mg/L, Harlan Laboratories Study D94990) showed that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2014: 0.36-1.1 mg/L).
The test method and the test species are recommended by the international test guidelines.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L
250 mg/L as CaCO3

Test temperature:

21°C

pH:

7.8 to 8.0

Dissolved oxygen:

8.2 to 8.7 Mg/L

Salinity:

CaCl2 × 2H2O: 294 mg/L
MgSO4 × 7H2O: 123 mg/L
NaHCO3: 65 mg/L
KCl: 5.8 mg/L

Nominal and measured concentrations:

At the start of the two renewal periods, the determined concentrations of the test item in the test media (dilution 1:460, 1:220, 1:100, 1:46, 1:22, 1:10 and 1:4.6) were 0.13, 0.29, 0.69, 1.5, 3.1, 6.9, 15 mg/L and 0.12, 0.26, 0.58, 1.3, 2.9, 6.4 mg/L, respectively. At the end of the test medium renewal periods the measured concentrations were 0.12, 0.25, 0.63, 1.3, 2.7, 5.6 and 12 mg/L and 0.11, 0.27, 0.57, 1.2, 2.7 and 6.1 mg/L.

Details on test conditions:

The test was performed in 100-mL glass beakers filled with 50 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.

A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 800 and 1060 Lux.

The daphnids were not fed during the test.

A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were transferred into clean test vessels with freshly prepared test medium of the corresponding concentration by wide-bore pipette.

The test concentrations were based on the results of a range-finding test and on the results of a pre-experiment to determine the solubility of the test item.

Biological results of the range-finding test (GLP) were as follows:

Control: 0% (48-hour immobilization)
1:100 Dilution: 10% (48-hour immobilization)
1:10 Dilution: 100% (48-hour immobilization)
Undiluted filtrate (Loading rate 100 mg/L): 100% (48-hour immobilization)

The following concentrations of test item were tested in the definitive test: dilutions 1:460, 1:220, 1:100, 1:46, 1:22, 1:10 and 1:4.6 of the undiluted filtrate with the loading rate of 100 mg/L. Additionally, a control was tested in parallel (test water without test item).

For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnid was 10 mL. The daphnids were randomly distributed to the test vessels at the initiation of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 1.3 mg/L. At the two next higher mean measured concentrations of 2.8 and 6.2 mg/L, the immobilization rates were 10 and 45%, respectively. Moreover, an adverse effect (reduced swimming activity) was recorded in 3 of the 11 remaining mobile test animals at the mean measured concentration of 6.2 mg/L. At the highest mean measured concentration of 13 mg/L, all test organisms were found to be immobile after 24 hours.
The 24-hour EC50 of CA5204A was calculated to be 6.3 mg/L with 95% confidence limits of 5.1 and 7.7 mg/L.
After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the mean measured concentration of 1.3 mg/L. At the test concentration of 2.8 mg/L, three test organisms were found to be immobile. Moreover, an adverse effect (reduced swimming activity) was recorded in 2 of 17 remaining mobile test animals at this test concentration. At the two highest mean measured concentrations of 6.2 and 13 mg/L, all test organisms were found to be immobile.
The 48-hour EC50 was calculated to be 3.9 mg/L with 95% confidence limits of 3.3 and 4.6 mg/L. The 48-hour NOEC of CA5204A was 1.3 mg/L, since no immobilization was observed up to and including this test concentration.
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the water surface).

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 was calculated to be 3.9 mg/L with 95% confidence limits of 3.3 and 4.6 mg/L. The 48-hour NOEC of CA5204A was 1.3 mg/L.

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004).

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared using ultrasonic treatment for 15 minutes and intense stirring by magnetic stirrers at room temperature in the dark over 3 hours.  

Following the stirring period, the dispersion was left to settle for 30 minutes. Thereafter, the middle phase of the dispersion was carefully separated from the non-dissolved test item and used as stock solution. The following dilutions were used as test media: 1:4.6, 1:10, 1:22, 1:46, 1:100, 1:220 and 1:460 of the stock solution with the loading rate of 100 mg/L. Additionally, a control was tested in parallel.

The preparation of the test media was based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

At the start of the two renewal periods, the determined concentrations of CA5204A in the test media (dilution 1:460, 1:220, 1:100, 1:46, 1:22, 1:10 and 1:4.6) were 0.13, 0.29, 0.69, 1.5, 3.1, 6.9, 15 mg/L and 0.12, 0.26, 0.58, 1.3, 2.9, 6.4 mg/L, respectively. At the end of the test medium renewal periods the measured concentrations were 0.12, 0.25, 0.63, 1.3, 2.7, 5.6 and 12 mg/L and 0.11, 0.27, 0.57, 1.2, 2.7 and 6.1 mg/L. From the dilution 1:4.6, the last samples were taken after 24 hours, since all daphnia were immobile at this concentration at this observation time.

The 48-hour EC50 was calculated to be 3.9 mg/L with 95% confidence limits of 3.3 and 4.6 mg/L. The 48-hour NOEC of CA5204A was 1.3 mg/L.

Description of key information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004).

The 48-hour EC50 was calculated to be 3.9 mg/L with 95% confidence limits of 3.3 and 4.6 mg/L. The 48-hour NOEC of CA5204A was 1.3 mg/L, since no immobilization was observed up to and including this test concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.9 mg/L