Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th May to 9th june 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Sponsor's information on this batch detailed in Notebook No. 02-181-126.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The corresponding time weighted mean measured concentrations were 0.015, 0.083, 0.365, 1.369 and 4.653 mg/L, which is equivalent to 30.0, 52.9, 73.3, 87.4 and 93.4 % of the nominal concentrations (corrected for purity). All results are based on time weighted means.

- Sampling method:
Duplicate samples were taken for chemical analysis at the beginning and end of 3 replacement periods over the duration of the test; these were on Days 0 and 3; 7 and 10; and 19 and 21 (for fresh and expired solutions respectively). Samples for fresh solutions were taken from the test solutions immediately after preparation. The expired samples were taken from a pooled sample of all replicates.
Test solutions were analysed according to the procedures established and validated under lnveresk Study No. 342753.

- Sample storage conditions before analysis:
Analyses were normally performed on the day of collection. In cases where this was not possible, samples were kept under suitable storage conditions until analysis took place.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Test solutions were prepared by parallel dilution of a 7 mg/L stock solution (the highest test concentration). This stock solution was prepared by adding ca 14 mg WRS-2390TX to Elendt M4 and bringing to volume in a 2 litre volumetric flask. The stock was then ultrasonicated for ca 10 min to aid dissolution.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: cultured within the laboratory by acyclical parthenogenesis.
- Age of parental stock (mean and range, SD): Neonates used were <24 h old at study initiation.
- Feeding during test
Daphnia were fed on a diet of concentrated green algae (Chlorella vulgaris) for both culturing and testing. During the reproduction test Daphnia were fed daily at a rate of ca 0.1-0.2 mg C/parent Daphnia/day.
To determine feeding rates for the test a nomograph of Total Organic Carbon (TOC) vs. cell numbers was prepared. This allowed the TOC content of a sample of concentrated Chlorella vulgaris to be determined from a background cell count.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

nda

Test conditions

Hardness:

The hardness and alkalinity of the Elendt M4 used to prepare the test solutions were in the range 216-240 mg CaCO3/L (hardness) and 60-95 mg CaCO3/L (alkalinity)

Test temperature:

19.2-22.3°C

pH:

7.26-7.92

Dissolved oxygen:

75.0-98.4 % ASV (air saturation value)

Salinity:

nda

Nominal and measured concentrations:

The corresponding time weighted mean measured concentrations were 0.015, 0.083, 0.365, 1.369 and 4.653 mg/L, which is equivalent to 30.0, 52.9, 73.3, 87.4 and 93.4 % of the nominal concentrations (corrected for purity). All results are based on time weighted means.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted using glass beakers (ca 100 mL capacity) covered with perspex lids to minimise dust contamination and evaporative losses. Prior to test initiation the beakers were acid washed and oven dried. Immediately prior to each use the beakers were rinsed with Elendt M4 medium.
Each vessel contained ca 60 mL of test or control media, as appropriate.
Test vessels were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate):
The test was conducted under semi-static conditions, with media replacement conducted three times weekly (Monday, Wednesday and Friday).
- No. of organisms per vessel: 10 (20 for control)
- No. of vessels per concentration (replicates): 10, the daphnia were housed singly for the duration of the test.
- No. of vessels per control (replicates): 20, the daphnia were housed singly for the duration of the test.


TEST MEDIUM / WATER PARAMETERS
Water quality parameters were measured at media change in a single replicate for each treatment group, prior to addition of the Daphnia (fresh media), and in expired media for all vessels.
Water quality parameters were measured using a Sentron Argus X pH meter and a YSI 550A dissolved oxygen meter.
Water quality parameters remained within the following range over the duration of the test: pH 7.26-7.92; temperature 19.2-22.3°C, dissolved oxygen 75.0-98.4 % ASV (air saturation value).
The hardness and alkalinity of the Elendt M4 used to prepare the test solutions were in the range 216-240 mg CaCO3/L (hardness) and 60-95 mg CaCO3/L (alkalinity).
Although the maximum temperature was briefly out-with acceptable limits this deviation did not have an impact on the outcome of the study


OTHER TEST CONDITIONS
- Photoperiod:
A light cycle of 16 h light and 8 h dark was in operation throughout the test. Artificial daylight fluorescent tubes provided illumination.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
All vessels were observed daily throughout the duration of the test for adult Daphnia health and neonate production. Neonates were discarded after counting. Males, where present, where discarded and discounted from the results.

Daphnia were recorded as immobile if no movement was observed within 15 s following gentle agitation of the test vessel.


RANGE-FINDING STUDY
Based on the results of an acute toxicity (EC50) test (conducted under Inveresk Study No. 803823) a reproduction test was conducted at 0.07, 0.22, 0.7, 2.2 and 7.0 mg/L WRS-2390TX, with an untreated control. These concentrations are equivalent to 0.050, 0.157, 0.498, 1.566 and 4.984 mgIL, corrected for test item purity (71.2%).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.365 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation and reproduction

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.69 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation and reproduction

Remarks on result:

other: As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, as such the estimated value should be regarded with caution and 95% confidence limits were not calculated.

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.365 - 0.69 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation and reproduction

Remarks on result:

other: All parental daphnids were dead by Days 14 and 21 at the highest two concentrations.

Details on results:

- Mortality of parent animals:
One daphnid each, from the control, 0.015 and 0.365 mg/L concentrations were observed to be immobilised during Days 7 to 10 during the test. No immobilisation was noted at the 0.083 mg/L concentration. All daphnids were immobilised on Day 14 for the 1.369 mg/L concentration, a day after the last reproductive cycle, combined test item and reproductive stress being the probable cause for the synchronised mortality. For the highest concentration of 4.653 mg/L, all daphnids were immobile by Day 4 (i.e. before reproduction started).
At test completion, 5.6, 14.3, 0, 16.7, 100 and 100 % immobilisation was observed in the control, 0.015, 0.083, 0.365, 1.369, and 4.653 mg/L treatment groups, respectively. The mortality for the parental daphnids was less than 20% by the end of the test for the control, 0.015, 0.083 and 0.365 mg/L treatment groups. Twenty per-cent is the maximum allowable control parental mortality stipulated by OECD Guideline 211, by inference it is therefore suggested that all mortality noted in test item treatments which is less than 20% can be regarded as natural susceptibility and not directly as an effect of the test item.

No significant decrease in reproduction was noted for all surviving treatment groups.

Where the sporadic appearance of males was noted in the treatment groups, they were treated in accordance with the following recommendations of OECD Guideline 211; "If, in any replicate the parent animal dies during the test or turns out to be male, then the replicate is excluded from the analysis. The analysis will be based on a reduced number of replicates". Hence males, when identified (smaller size and more intense colouration), were discarded and discounted from the test results.

By Day 7 reproduction had been noted in all treatments, excluding the highest concentration of 4.653 mg/L, where all daphnids were dead. The coefficient of variation is greatest in the highest surviving concentration of 1.369 mglL, probably as a consequence of the majority of neonates first appearing on Day 8. This suggests that reproduction was delayed in this concentration, this however has limited relevance as all parental daphnids were dead by Day 14. From the Day 7 data, the EC50 for WRS-2390TX to Daphnia reproduction was estimated as 0.69 mg/L. As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, as such the estimated value should be regarded with caution and 95% confidence limits were not calculated.

As all parent daphnids were dead by Days 14 and 21 at the highest two concentrations and no reproduction effect was noted at 0.365 mg/L, the EC50 for these time points was estimated to be between 0.365 and 0.69 mg/L (i.e. between the NOEC and Day 7 EC50).
The Day 21 No Observed Effect Concentration (NOEC) for WRS-2390TX to Daphnia reproduction was 0.365 mg/L, under the conditions of the test.

Results with reference substance (positive control):

nda

Reported statistics and error estimates:

- Data from male replicates were excluded from the statistical analyses. Additionally, data from replicates that were not alive at the timepoint of interest were excluded from the statistical analyses at that timepoint.
- At Day 7, using Wadleys adjustment, the Pearson's chi-square test statistic was significant at the 1% level (p=0.007), therefore the estimate of the EC50 of 0.69 mg/L should be treated with caution and no confidence intervals have been reported. At Day 14 and 21, using Wadleys adjustment, the data did not span 50%, and as a result it was not possible to estimate the EC50 values at these timepoints.
- For the estimate of the NOEC, at all timepoints, the homogeneity of variance assumption was satisfied at the 1% significance level using Levene's test, therefore all analyses were performed using untransformed data. The estimate of the NOEC was 0.365 mg/L for Day 7 and Day 14. For Day 21, there were no data for the 4.653 mg/L dose level, and there were no statistically significant decreases (versus control) at all other dose levels; therefore the estimate of the NOEC was 0.365 mglL for Day 21.

Any other information on results incl. tables

All prepared test solutions were clear and colourless at preparation.

One daphnid each, from the control, 0.015 and 0.365 mg/L concentrations were observed to be immobilised during Days 7 to 10 during the test. No immobilisation was noted at the 0.083 mg/L concentration. All daphnids were immobilised on Day 14 for the 1.369 mg/L concentration, a day after the last reproductive cycle, combined test item and reproductive stress being the probable cause for the synchronised mortality. For the highest concentration of 4.653 mg/L, all daphnids were immobile by Day 4 (i.e. before reproduction started).

At test completion, 5.6, 14.3, 0, 16.7, 100 and 100 % immobilisation was observed in the control, 0.015, 0.083, 0.365, 1.369, and 4.653 mg/L treatment groups, respectively. The mortality for the parental daphnids was less than 20% by the end of the test for the control, 0.015, 0.083 and 0.365 mg/L treatment groups. Twenty per-cent is the maximum allowable control parental mortality stipulated by OECD Guideline 211, by inference it is therefore suggested that all mortality noted in test item treatments which is less than 20% can be regarded as natural susceptibility and not directly as an effect of the test item.

Summary of Daphnia Reproduction:

Time Weighted Mean Concentration of WRS-2390TX (mg/L)	Mean Days to First Brood	Mean Number of Neonates Produced after 7 Days	Mean Number of Neonates Produced after 14 Days	Mean Number of Neonates Produced after 21 Days
Control	7.5	14 [38.8]	88  [52]	146  [6.3]
0.015	7.5	13 [71.2]	88 [10.5]	151 [14.8]
0 . 083	8.5	17 [41.0]	97  [7.0]	173  [8.3]
0 . 365	8 . 5	9 [77.8]	106  [7.1]	213  [9.0]
1.369	8.5	6 [155 .0]	NA	NA
4.653	NA	NA	NA	NA

NA = Not applicable, all dead

[Coefficient of variation %]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The Day 7 EC50 for WRS-2390TX to Daphnia reproduction was estimated as 0.69 mg/L. As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, as such the estimated value should be regarded with caution and 95% confidence limits were not calculated.
As all parental daphnids were dead by Days 14 and 21 at the highest two concentrations and no reproduction effect was noted at 0.365 mg/L, the EC50 for these time points was estimated to be between 0.365 and 0.69 mg/L (ie between the NOEC and Day 7 EC50).
The Day 21 No Observed Effect Concentration (NOEC) for WRS-2390TX to Daphnia reproduction was 0.365 mg/L, under the conditions of the test.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In a Daphnia Reproduction Test (21 Day, Semi-Static) (Inveresk report number: 24048) WRS-2390TX was found to have an NOEC of 0.365 mg/L. The Day 7 EC50 for WRS-2390TX to Daphnia reproduction was estimated as 0.69 mg/L. As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, as such the estimated value should be regarded with caution and 95% confidence limits were not calculated.

As all parental daphnids were dead by Days 14 and 21 at the highest two concentrations and no reproduction effect was noted at 0.365 mg/L, the EC50 for these time points was estimated to be between 0.365 and 0.69 mg/L (ie between the NOEC and Day 7 EC50)

By Day 7 reproduction had been noted in all treatments, excluding the highest concentration of 4.653 mg/L, where all daphnids were dead. The coefficient of variation is greatest in the highest surviving concentration of 1.369 mglL, probably as a consequence of the majority of neonates first appearing on Day 8. This suggests that reproduction was delayed in this concentration, this however has limited relevance as all parental daphnids were dead by Day 14. From the Day 7 data, the EC50 for WRS-2390TX to Daphnia reproduction was estimated as 0.69 mg/L. As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, as such the estimated value should be regarded with caution and 95% confidence limits were not calculated.

The study was conducted under semi-static conditions in accordance with OECD (1998) Guideline 211 and EC (2001) Method C20 and is in compliance with the OECD principles of Good Laboratory Practice.