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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Remarks:

The fish embryo and Sac-fry acute toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2015 to 11 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples (ca 10 or 20 mL) of the control and each test concentration were taken for analysis of freshly prepared test media on Days 0, 3 (pre-hatch) and Day 2 (post-hatch). The corresponding 24-hour old media was analysed on Days 1, 4 (pre-hatch) and Day 3 (post-hatch).
At each sampling occasion, duplicate samples were taken. One for chemical analysis and one as a ‘back up’ sample.

Vehicle:

yes

Details on test solutions:

The dilution water used for conducting the tests was treated mains water that had been passed through activated carbon filters.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Species: Fathead minnow (Pimephales promelas)
Source: Fertilised eggs were obtained from in house laboratory
Egg fertility of batch: 92%, demonstrating the eggs were of a suitable quality for the test.
Acclimatisation period: None
Treatment for disease: None
Feeding: fed ad libitum using freshly hatched brine shrimp (Artemia salinis) nauplii and also with Tetramin® flake food

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 h

Hardness:

84 to 87 mg/L as CaCO3

Test temperature:

22.5 to 25.7ºC

pH:

7.25 to 7.70

Dissolved oxygen:

72 to 103%

Nominal and measured concentrations:

The definitive test was conducted at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% of the saturated solution (0.11, 0.40, 1.3, 4.0 and 14 mg/L based on time-weighted mean measured concentrations).

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL crystalising dishes.
- Aeration: Gentle
- Renewal rate of test solution (frequency/flow rate):
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


OTHER TEST CONDITIONS

- Photoperiod: 16 hours fluorescent light and 8 hours dark


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations for hatching success, larval mortality, deformed larvae and other symptoms of toxicity were made daily during the pre and post-hatch phases, as appropriate. At the end of the test, lengths and dry weights of the surviving fry were measured.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Nominal concentrations of 0.10, 1.0, 10 and 100% of the saturated solution.
- Results used to determine the conditions for the definitive study: No effects were observed on hatching success or post-hatch survival up to the 1.0% saturated solution test group (up to 4 days post-hatch). At a concentration of 10% saturated solution, hatching success appeared unaffected however no surviving larvae remained at Day 4 post-hatch. No eggs hatched at the 100% saturated solution test concentration.

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Hatching success - Statistical analysis of the hatching success data showed a significant difference between the control and 14 mg/L test concentration. There were no statistically significant effects on hatching success at any of the remaining test concentrations compared to the control. The NOEC and LOEC values for hatching success were considered to be 4.0 and 14 mg/L, respectively. The LC50 value for hatching success was estimated to be 6.3 mg/L.

Post-hatch survival - There were no statistically significant effects on post-hatch survival at the 0.11 to 1.3 mg/L test concentrations compared to the control. A statistically significant effect was observed between the 4.0 mg/L and the control in terms of post-hatch survival. The NOEC and LOEC values for post-hatch survival were considered to be 1.3 and 4.0 mg/L, respectively. The LC50 value for post-hatch survival was estimated to be 3.4 mg/L.

Growth - A statistically significant effect was observed at the 1.3 and 4.0 mg/L test concentrations, in terms of total lengths, compared to the control. The NOEC and LOEC values for total length were considered to be 0.40 and 1.3 mg/L, respectively. Although fish survived at the 4.0 mg/L concentration, the number and consequently the weight were too small for an accurate measurement to be determined. Based on visual inspection of the data the NOEC and LOEC values were considered to be 0.11 and 0.40 mg/L respectively.

Validity criteria fulfilled:

yes

Conclusions:

Toxicity endpoints were identified for CA5204A to early life stages of Fathead minnow (Pimephales promelas).The overall LC50, LOEC and NOEC concentrations based on all endpoints, were considered to be 3.4, 0.40 and 0.11 mg/L, respectively.

Executive summary:

The objective of the study was to determine the lethal and sub-lethal effects of the test substance CA5204A on the early development and juvenile stages of the freshwater fish species Pimephales promelas, in accordance with OECD Chemical test Guideline No. 212 Fish, Shortterm ToxicityTest on Embryo and sac-fry Stages.

Due to the solubility/stabilityof the CA5204A the test was conducted using semi-static test conditions with dailymedia renewals throughout the test. CA5204A was prepared as a saturated solution. An excess of CA5204A (100 mg/L) was added to treated mains water, sonicated for ca 15 minutes and vigorously stirred for ca 1 hour after which the preparation was allowed to settle for ca 30 minutes. The aqueous phase was then removed by syphon and filtered through a 0.45 µm filter to give the 100% saturated solution. Dilutions were then prepared, in treated mains water, from the 100% saturated solution to give the test concentrations.

Based on the results of a range-finding test, the definitive test was conducted at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% of the saturated solution (0.11, 0.40, 1.3, 4.0 and 14 mg/L based on time-weighted mean measured concentrations). Three replicate test vessels were prepared for each concentration and control group.

At the start of the test, ten apparently viable eggs from in-house brood fish, were added to each test vessel. The organisms were observed daily.

Analysis of the test media samples, for CA5204A, was conducted on the freshly prepared test media on Days 0, 3 (pre-hatch) and Day 2 (post-hatch). The corresponding 24-hour old media was analysed on Days 1, 4 (pre-hatch) and Day 3 (post-hatch).

The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were determined for hatching success, post-hatch survival and growth (based on length and dry weight). Where applicable, the LC50 values were also determined.

Chemical analysis of the freshly prepared media showed measured concentrations of CA5204A ranged from 0.120 to 15 mg/L. Analysis of the corresponding 24-hour old media samples showed measured concentrations to range from 0.0857 to 13 mg/L. Given the decline in measured concentration observed over each 24-hour period, the results have been calculated based on the time-weighted mean measured concentrations. These were calculated to be 0.11, 0.40, 1.3, 4.0 and 14 mg/L for the 0.32, 1.0, 3.2, 10 and 32% saturated solution test concentrations, respectively.

The mean hatching success of embryos in the control vessels was 97%, which was in excess of the validity criterion of ≥70%.  The post-hatch survival in the pooled control vessels was 97%, which was greater than the validity criterion of ≥75%.  The test is therefore considered to be valid.

The overall LC50, LOEC and NOEC concentrations based on all endpoints, were considered to be 3.4, 0.40 and 0.11 mg/L, respectively.

Description of key information

Toxicity endpoints were identified for CA5204A to early life stages of Fathead minnow (Pimephales promelas).The overall LC50, LOEC and NOEC concentrations based on all endpoints, were considered to be 3.4, 0.40 and 0.11 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.11 mg/L