Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

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:

14. November 2013 - 02. April 2014

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

(1)Calculation error - three lowest test concentrations. Results not affected by deviation. (2)Light intensity was measured before and after the test. No influenece on integrity. (3)Temparture range above 22°C - no effect on integrity

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

other: yellowish-brown liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations:
Different concentrations were dissolved at the test medium according Elendt M4 given in the guideline OECD211.
The main test was performed using nominal concentrations of 0.020*), 0.047**), 0.113***), 0,39, and 0.90 mg/L.
*) instead of 0.03mg/L planned for geom. series (separation factor 2.3) modification due to calculation error
**) instead of 0.07mg/L planned for geom. series (separation factor 2.3) modification due to calculation error
***) instead of 0.17mg/L planned for geom. series (separation factor 2.3) modification due to calculation error
The modified concentration steps did not affect either the results of the test or the integrity of the study.

Sampling method:
The application method was performed according to the OECD guideline 211.
All treatments are renewed daily in order to assure that the animals were exposed to the test item at almost the same concentrations.
Preparation of a stock solution by using dilution water. The stock solutions with the macronutrients were individually prepared in ultrapure water (for each salt a separate solution). From these individual stock solution, the test solution was prepared using algal-supension, stock solution, and Elendt M4 medium in specific amounts to a total of 1500ml.

Sample storage conditions before analysis:
As the supporting analyse could not be performed on the same day the collection of the specimens. Therefore the specimen to be analysed were deep-frozen stored in glass flasks until the day of the analyses. Specimens were colected from freshly prepared test solutions and from those incubated with the daphnia for 24hr. Within the study, three pairs of specimens (freshly prepared and 24hr incubated) were analysed for each concentration.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation and solution of the test item
- The stock solution of the test item was prepared by solving the test item (10mg in 2000ml)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Organism
Daphnia magna STRAUS (clone 5)
Laboratory bred (derived from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique Appliquee, France in 1978 and cultivated since then at BASF using medium M4 acc. Elendt)
The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium (see below) at an interval of 1 to 3 days followed by feeding with Desmodesmus subspicatus- and/or Chlorella-cells. Daphnia used for the test are not older than 24 h. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 h.

Acclimation
The daphnia (parent animals) are held in the test medium (M4 according Elendt) and thus had not to be adapted to that standard medium.
The animals were fed daily with unicellular cells of Desmodesmus subspicatus.
The amount of the algae was calculated to refer to a content of 0.1-0.2 mg C / Daphnia (corresponding to 1-2 mg C/L).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no observation

Test conditions

Hardness:

see 8.3.1.1 ff

Test temperature:

see table 7

pH:

see table 7

Dissolved oxygen:

see table 7

Nominal and measured concentrations:

see table 8

Details on test conditions:

The objective of the test was to assess the effect of the test item on the reproductive output of Daphnia magna. For this purpose, young female Daphnia (the parent animals), aged less than 24 h at the start of the test, were exposed to 5 concentrations of the test item added to the test medium. The test duration was 21 days. At the end of the test, the total number of living offspring produced per parent animal alive at the end was assessed. This means, that juveniles produced by adults that died during the test were excluded from the calculations. The reproductive output of the animals exposed to the test item was compared to that of the control in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC). In addition, the EC10, EC20 and EC50 were calculated. Besides that, the survival of the parent animals and time to production of the first brood are reported. Furthermore, the size of the daphnia at the end of the test was determined in order to investigate side-effects on growth.
Daphnia magna STRAUS (clone 5) of laboratory bred (derived from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique Appliquee, France in 1978 and cultivated since then at BASF using medium M4 acc. Elendt) were used for the test. In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this bred was tested prior to beginning of the test. The value of the EC50 (24 h) in this period of time was 1.06 mg/L potassium dichromate. This value is in accordance with the recommendations given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test).
The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium (see below) at an interval of 1 to 3 days followed by feeding with Desmodesmus subspicatus- and/or Chlorella-cells. Daphnia used for the test are not older than 24 h. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24 h.
The daphnia (parent animals) are held in the test medium (M4 according Elendt) and thus had not to be adapted to that standard medium. At the beginning of the test, the new born daphnia being not older than 24 h were transferred individually into the different test vessels (beakers) containing approximately 90 mL of the treatments (one animal each vessel). The test item was introduced as a stock solution prepared in the medium according Elendt M4. The concentration range was fixed together with the Sponsor considering two acute daphnia toxicity tests with EC50 values (acute) being in the range of 5-9 mg/L. The following nominal concentrations were applied in the main test: 0.0 mg/L (control), of 0.020, 0.047, 0.113, 0.39, and 0.90 mg/L. For each concentration, 13 parallels were prepared. The animals were fed with unicellular cells of Desmodesmus subspicatus. The amount of the algae was calculated to refer to a content of 0.1-0.2 mg C / Daphnia (corresponding to 1-2 mg C/L). The test was performed semistatically; this means that the daphnia were transferred into freshly prepared treatments every day. As supporting analyses, pH and oxygen content of the test solutions were measured in the freshly prepared test solutions and in those being incubated for a period of 24 h. The incubation took place in beakers filled with 90 mL of the treatments, respectively. All values were documented in data sheets.The test duration was 21 days. The temperature range in the test room was shown to be at 20.1 – 23.1 °C but temperatures in thetreatments measured were in the range of 20.0 – 22.0. There was a day-night-period of 16hr/8hr. With the exception of one lamp, all lamps were switched off in the test room after daily manipulation in order to get a low light intensity. The mean light intensity in the test room was between 0.56 and 0.66 µmol*m2*s-1 (0.56 - 0.66 klx) corresponding to ≈ 9-10 µE/m2s (measured before and after the test period), and thus in accordance with OECD 211 not exceeding 15 - 20 µE/m2s. Daily the test solutions were investigated for appearance of offspring animals, and the numbers were recorded. The newborn offspring animals were eliminated daily from the treatments. For this purpose, the whole contents of test vessels were transferred into a large glass-petri dish which facilitated the manipulation. The daphnia could then be easily picked up and transferred into the new prepared test solutions, and newborne offspring daphnia could be counted.

During the study, the numbers of living offspring animals were determined with each parent animal, and the numbers from each concentration were set into relation with those of the control without any test item. The aim of the test is to determine the so-called LOEC („Lowest Observed Effect Concentration“), and the NOEC („No Observed Effect Concentration”). Additionally the EC10, EC20 and EC50 should be determined.

At the end of the test, the size of the parent daphnia was measured using a binocular microscope equipped with a digital camera. Using scales below the daphnia, the diameters of all daphnia of each the control and the treatments with the test item were determined. This parameter is used as an additional growth parameter for the evaluation of the toxicity effect.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not apllicable

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.379 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not applicable

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not applicable

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not applicable

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.604 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - 0.604-0.604

Effect concentrations 6

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.574 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - not given due to mathematical reasons

Effect concentrations 7

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

0.718 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - 0.718 - 0.719

Effect concentrations 8

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

0.678 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - not given due to mathematical reasons

Effect concentrations 9

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.001 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - 1.001-1.001

Effect concentrations 10

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% confidence level) - not given due to mathematical reasons

Details on results:

Mortality of parent animals after 7, 14, and 21 days:
see table 3 (report)
Number of offspring produced per day per female from day 9 to 21:
see table 6 (report)
Body length and weight of parent animals:
see tables 10.3ff (report) Additional Size-Measurements of the Daphnia after Incubation
Type and number of morphological abnormalities:
no abnormalties discovered
Type and number of behavioural abnormalities:
no abnormalties discovered
Number of females (parental):
72 femal animals for all groups (1 group 6 animals was arithmetically eliminated)
Time to first brood release or time to hatch
Control: 11.5 (day/mean)
Nominal concentration (0.02) : 10.8 (day/mean)
Nominal concentration (0.047): 11.0 (day/mean)
Nominal concentration (0.113): 10.7 (day/mean)
Nominal concentration (0.390): 10.8 (day/mean)
Nominal concentration (0.900): 13.4 (day/mean)
No relevant biochemical changes or biological observations

Results with reference substance (positive control):

no reference substance used

Reported statistics and error estimates:

Parameters analysed:
- Mean cumulative offspring per survivor in daphnia magna
- Mobility
- Age of first reproduction
- Intrinsic rate
Statistical methods:
- Probit analysis (ToxRatPro version 2.10)
Probit slopes:
See tables 6 / 15 / 26 in apendices report for parameters of the probit analysis

Error estimates (confidence interval 95%)

Any other information on results incl. tables

Report

Table 3: Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 12)

Nominal Concentration [mg/L]	Mortality [%]
	7 days	14 days	21 days
0.020	0	0	0
0.047	0	0	1
0.113	0	0	2
0.390	0	0	0
0.900	1	4	5
Control	0	0	0
EC50 adult mortality	>0.9 mg/L

Table 6:         Detailed Results on Reproduction and Mortalities

Nominal Concentration:0 (Control)
Day^1)		10	11	12	13	14	15	16	17	18	19	20	21
Vessel No.	No. of offspring Animals													Total alive
1		0	9	0	0	20	0	30	0	0	23	0	0	82
2		0	8	0	0	18	0	0	29	0	0	23	0	76
3		0	6	0	19	0	0	26	0	0	23	0	0	74
4		0	5	0	7	10	0	27	1	0	26	0	0	76
5		0	8	0	15	8	0	28	2	0	0	16	0	(77)*^)
6		0	9	0	0	21	0	0	27	1	0	26	0	84
7		0	6	0	0	19	0	0	29	0	0	23	0	77
8		0	8	0	24	0	0	32	0	0	12	11	0	87
9		0	9	0	0	19	0	0	28	0	0	22	0	78
10		0	6	0	0	18	0	0	33	0	0	30	0	87
11		0	0	12	0	0	22	0	0	25	0	0	20	79
12		0	7	0	0	18	0	0	27	0	0	22	0	74
13		12	0	0	18	0	0	26	1	0	19	0	0	76
Dead parent animal			0	0	0	0	0	0	0	0	0	0	0	-

¹⁾Day 1-9 no offspring animals were observed 

*⁾Results with animal No. 5 was statistically eliminated as one animal had been killed upon manipulation during the test within concentration step 0.02 mg/L representing immobilisation not due to effects of the test item. Therefore, automatic routine analysis was not possible without special measures. For this reason, using the random generator in EXCEL, following twelve vessels were chosen for routine statistical analysis with the ToxRat Program: No. 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, and for all other test concentrations, also 12 animals are used for statistical evaluation.

Other concentrations and tables (0.020 mg/L; 0.047 mg/L ; 0.113 mg/L; 0.39 mg/L; 0.900mg/L) to be found in the attached report.

10.3.1 Body Length

Replicate No.	Nominal Concentration (mg/L)
	Control	0.020	0.047	0.113	0.39	0.90
1	4.27	--*^)	4.58	4.53	4.12	3.74
2	4.12	4.17	4.22	4.40	4.45	3.92
3	4.12	4.19	4.40	4.25	4.12	3.97
4	4.12	4.45	4.25	M	4.45	3.51
5	--*^)	4.40	M	4.14	4.19	M
6	4.47	4.45	4.45	4.53	4.55	M
7	4.14	4.22	4.12	4.53	4.45	3.94
8	4.50	4.58	4.53	4.53	--*^)	4.02
9	4.47	4.22	4.25	M	4.22	3.94
10	4.12	4.27	--*^)	4.12	4.25	3.84
11	4.42	4.30	4.17	4.32	4.07	--*^)
12	4.04	4.60	4.32	--*^)	4.45	M
13	4.45	4.22	4.40	4.42	4.09	M
No. of Replicates	12	12	11	10	12	8
Mean	4.27	4.34	4.34	4.38	4.28	3.86
Std.Dev	0.18	0.15	0.15	0.16	0.17	0.17
CV%	4.2	3.5	3.4	3.7	4.1	4.3

 

10.3.2 Body Width

Replicate No.	Nominal Concentration (mg/L)
	Control	0.020	0.047	0.113	0.39	0.90
1	2.80	--*^)	2.77	2.75	2.62	2.52
2	2.54	2.67	2.80	2.72	2.72	2.44
3	2.67	2.72	2.85	2.75	2.67	2.54
4	2.72	2.69	2.75	M	2.69	2.34
5	--*^)	2.80	M	2.62	2.72	M
6	2.72	2.82	2.77	2.77	2.77	M
7	2.69	2.64	2.75	2.67	2.72	2.34
8	2.75	2.75	2.77	2.77	--*^)	2.44
9	2.72	2.62	2.82	M	2.54	2.42
10	2.75	2.72	--*^)	2.75	2.67	2.42
11	2.69	2.77	2.69	2.69	2.62	--*^)
12	2.72	2.75	2.64	--*^)	2.67	M
13	2.69	2.64	2.67	2.75	2.59	M
No. of Replicates	12	12	11	10	12	8
Mean	2.71	2.72	2.75	2.72	2.67	2.43
Std.Dev	0.06	0.07	0.06	0.05	0.06	0.07
CV%	2.3	2.4	2.3	1.8	2.4	3.0

--*⁾Replicate was statistically excluded from statistical treatments as one animal had been killed upon manipulation during the test within concentration step 0.02 mg/L representing immobilisation not due to effects of the test item. Therefore, automatic routine analysis was not possible without special measures. For this reason, using the random generator in EXCEL, the indicated twelve vessels were chosen for routine statistical analysis with the ToxRat Program.

Measured Concentration (Appendix)

Parameters of the probit analysis (Offspring per survivor)

Tab. 6: Parameters of the probit analysis: Results of the regression analysis

                            Parameter                                Value

                            Computation runs:                           9

                            Slope b:                                6,12452

                            Intercept a:                            0,19324

                            Variance of b:                 2.543,09619

                            Goodness of Fit                                

                            Chi²:                                      0,00000

                            Degrees of freedom:                       3

                            p(Chi²):                                        n.d.

                            Log EC50:                            -0,03155

                            SE Log EC50:                       0,41021

                            g-Criterion:                            0,00000

                            Residual Variance (Chi²/df):    0,00000

                            r²:                                             1,000

                            F:                                  -5279104,000

                            p(F) (df: 1;3):                               n.d.

Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0,100, data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (= are made wider; so, check whether these results are reasonable!). The coefficient of determination, r² (0 <= r² <= 1), gives the proportion of variance explained by the dose/response function.F-Test for regression: Ho: Slope = 0; hence, if p(F) <= alpha, the selected significance level, (e.g., alpha = 0.05) the regression revealed significant results (= slope is significantly different from zero).

Parameters of the probit analysis (Mobility)

Tab. 15: Parameters of the probit analysis: Results of the regression analysis

                            Parameter                                Value

                            Computation runs:                           6

                            Slope b:                                0,69515

                            Intercept a:                           -0,65965

                            Variance of b:                        0,15630

                            Goodness of Fit                                

                            Chi²:                                      4,74704

                            Degrees of freedom:                       3

                            p(Chi²):                                  0,19129

                            Log EC50:                             0,94894

                            SE Log EC50:                       0,98073

                            g-Criterion:                            1,24255

                            F:                                             1,954

                            p(F) (df: 1;3):                            0,257

Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0,100, data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (= are made wider; so, check whether these results are reasonable!).

 

No meaningful concentration/response was found (p(F) > 0.05; i.e. slope of the relationship is not significant different from zero).

Due to the lacking concentration/response the shown ECx are not valid.

Parameters of the probit analysis (Age at first reproduction)

Tab. 26: Parameters of the probit analysis: Results of the regression analysis

                            Parameter                                Value

                            Computation runs:                         13

                            Slope b:                              11,86428

                            Intercept a:                            0,30479

                            Variance of b:     1.704.233.984,00000

                            Goodness of Fit                                

                            Chi²:                                      0,00000

                            Degrees of freedom:                       3

                            p(Chi²):                                  1,00000

                            Log EC50:                            -0,02569

                            SE Log EC50:                    174,08523

                            g-Criterion:                            0,65302

                            Residual Variance (Chi²/df):    0,00000

                            r²:                                             0,838

                            F:                                           15,505

                            p(F) (df: 1;3):                            0,029

Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0,100, data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (= are made wider; so, check whether these results are reasonable!). The coefficient of determination, r² (0 <= r² <= 1), gives the proportion of variance explained by the dose/response function.F-Test for regression: Ho: Slope = 0; hence, if p(F) <= alpha, the selected significance level, (e.g., alpha = 0.05) the regression revealed significant results (= slope is significantly different from zero).

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

see executive summary

Executive summary:

Product LE 097 was tested for its effects on the reproductive output of Daphnia magna according to OECD-Test Guideline 211. For this purpose, young female Daphnia (the parent animals) aged less than 24h at the start of the test were exposed to the test item at five concentrations. The test duration was 21 days. Within that time cumulative living offspring produced per parent animal was recorded for each concentration. The reproductive output of the animals exposed to the different concentrations of the test item was compared to that of the controls in order to determine the lowest observed effect concentration (LOEC) and the no observed effect concentration of the test item (NOEC). Besides these two parameters, the EC₁₀, EC₂₀and EC₅₀were calculated. The following effect concentrations were found:

 

Mean Cumulative Offspring of Survivors
	Basis: Nominal Concentrations			Basis: Measured Concentrations
NOEC LOEC EC10 EC20 EC50	= = = = =	0.390 mg/L 0.900 mg/L 0.604 mg/L 0.718 mg/L 1.001 mg/L	(0.604 - 0.604)^1) (0.718 - 0.719)^1) (1.001 - 1.001)^1)	= = = = =		0.379 mg/L 0.840 mg/L 0.574 mg/L 0.678 mg/L 0.930 mg/L	[n.d.^1)]^2) [n.d.^1)]^2) [n.d.^1)]^2)
Age of first Reproduction
	Basis: Nominal Concentrations			Basis: Measured Concentrations
NOEC LOEC	= =	0.390 mg/L 0.900 mg/L		= =		0.379 mg/L 0.840 mg/L
Intrinsic Rate
NOEC LOEC EC10 EC20 EC50	= = = = =	0.390 mg/L 0.900 mg/L 0.783 mg/L 0.856 mg/L 1.015 mg/L	[(n.d.)^1)]^2) [(n.d.)^1)]^2) [(n.d.)^1)]^2)	= = = = =	0.379 mg/L 0.840 mg/L 0.735 mg/L 0.801 mg/L 0.943 mg/L		(0.419 - 0.780)^1) (0.654 - 0.818)^1) (0.895 - 1.531)^1)
Mobility / Mortality
	Basis: Nominal Concentrations			Basis: Measured Concentrations
NOEC LOEC EC10 EC20 EC50	≥ >  = = =	0.900 mg/L 0.900 mg/L 0.139 mg/L 0.563 mg/L n.d.^2)	[(n.d.)^1)]^2) [(n.d.)^1)]^2) (>0.9 mg/L)	≥ >  = = =	0.840 mg/L 0.840 mg/L 0.127 mg/L 0.547 mg/L n.d.^2)		[(n.d.)^1)]^2) [(n.d.)^1)]^2) (>0.840 mg/L)

¹⁾Numbers in brackets indicate the lower and upper confidence limits at the 95% confidence level.

²⁾No values are given due to mathematical reasons.

Concerning “mobility/mortality” statistical calculations have to be regarded as follows: at the nominal concentration step 0.9 mg/L, 5 animals were immobilized. At the concentration step lower (0.39 mg/L) there wasnomortality, and therefore the immobilization at the lower concentrations than 0.39 mg/L are not considered as such (EC₁₀and EC₂₀values are calculated by the computer program – but these are obviously extrapolated values calculated from probit analyses and are not considered significant). Therefore, NOEC for “mobility / mortality” was considered to be 0.84 mg/L (basis: measured concentration).

 

Test item related effects were found for the additional endpoint size length and width of animals: There is statistical evidence between the control and the test item. A NOEC of 0.390 mg/L (nominal concentration) was calculated for body length as well as for the body width. The LOEC was recogniced as being 0.9 mg/L (nominal concentration) for both parameters body length and body width. EC₅₀for body length was calculated to be 1.28 mg/L (nominal concentration) and >1.34 mg/L for body width (nominal concentration).

Within chemical analyses performed with the test solutions at the start of incubation (three times during the test) and after 24 hr, respectively, it could be shown that the test item remained always stable at the requested level of ≥ 80% in the aqueous phase.Nevertheless,the measured concentrations of Product LE 097 within the treatments deviated partially more than 20 % from the nominal values.For this reason, the results of this test should be based on the measured concentrations tested.