Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June - 06 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples Nominally ≤ 160 μg/L
An accurate volume of test solution was transferred, using glass pipettes into glass vessels and accurately fortified (if required). The solutions were then diluted, using glass pipettes into glass vessels, ×2 with 0.2% acetic acid in LC-MS methanol. The solutions were further diluted, at least ×2 with 0.1:50:50 acetic acid:water:methanol (v/v/v) and aliquot(s) taken for analysis by LC-MS/MS.
Excess sample dilutions were stored refrigerated.
Samples Nominally > 160 μg/L
An accurate volume of test solution was transferred, using glass pipettes into glass vessels and accurately fortified (if required). The solutions were then diluted, using glass pipettes into glass vessels, ×2 with 0.2% acetic acid in LC-MS methanol and further diluted (as required, minimum of ×2) with 0.1:50:50 acetic acid:water:methanol (v/v/v) to within the calibration range. Aliquot(s) of the diluted solution(s) were then taken for analysis by LC-MS/MS.
Excess sample dilutions were stored refrigerated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
This study was run with a dilution water control and nominal exposure concentrations of 0.09, 0.20, 0.43, 0.90 and 2.0 mg a.i./L.
A sub-sample of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts, was initially heated for approximately 30 minutes at 35°C then stirred with a glass pipette prior to weighing.
A nominal concentration of 200 mg a.i./L (based on 39% purity value), was prepared by adding a nominal 0.513 g of test substance to 1000 mL of D. magna media in a volumetric flask.
The primary stock was stirred on a magnetic stirrer with a magnetic PTFE follower for approximately 5 minutes. The primary stock was observed to be a clear and colourless solution.
The primary stock (PS1) of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts was then used to prepare the intermediate stock (ISI). 100 mL of primary stock was added to a final volume of 1000 mL of D. magna media in a volumetric flask. This intermediate stock was then used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of intermediate stock to 1000 mL of D. magna media (detailed in the following table).

Nominal concentration Intermediate stock concentration Volume prepared Volume of intermediate stock added Stock reference
(mg a.i./L) (mg a.i./L) (mL) (mL)
Control N/A 1000 N/A N/A
0.09 20 1000 4.5 IS1
0.20 20 1000 10 IS1
0.43 20 1000 21.5 IS1
0.9 20 1000 45 IS1
2.0 20 1000 100 IS1

All solutions were stirred on a magnetic stirrer with a magnetic PTFE follower prior to use.
All test solutions were observed to be clear and colourless prior to use.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Clone A, originally sourced from INRAE in France, obtained from continuous laboratory cultures held at Scymaris.

The D. magna cultures were fed on a mixed diet of YCT (yeast cereal trout) and Pseudokirchneriella subcapitata (now known as Raphidocelis subcapitata), strain CCAP 278/4.
The D. magna cultures were fed daily a prescribed diet depending on age and density of the culture.
Culture conditions were such that the D. magna reproduction was by diploid parthenogenesis.

D. magna <24 hours old and not first brood progeny were obtained from one culture vessel and were used for testing.
The parent animals were 12 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium since birth.
The test organisms were obtained from healthy cultures with no evidence of stress such as high mortality, presence of males or ephippia, delays in the production of the first brood or discoloured animals before the test period.
Test organisms were fed a mixed diet (YCT and P. subcapitata) approximately 180 minutes before the start of the test to minimise potential starvation effects during the pre-test holding period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The hardness (as CaCO3) measured ranged from 249 to 275 mg/L (Table 9, attached)

Test temperature:

Temperatures recorded daily over the 21 days ranged from 20.1 to 22.4°C with individual data shown in Table 10, attached. Although the temperature was measured to be outside 21 ± 1°C on Day 2, given that were no significant effects observed in the controls or any treatment during the study, this deviation to the study plan and guideline is considered to have no impact on the outcome or validity of the test.

pH:

The pH measured ranged from 7.48 to 8.17. (Table 8, attached)

Dissolved oxygen:

The dissolved oxygen concentrations measured ranged from 6.95 to 8.85 mg/L. (Table 7, attached)

Nominal and measured concentrations:

Nominal concentrations (based on registered substance): dilution water control and 0.09, 0.20, 0.43, 0.90 and 2.0 mg a.i./L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers with loose fitting lids
- Material, size, headspace, fill volume: filled with approximately 80 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Every 24 hours. On each renewal day, new test solutions were prepared and dispensed to a second set of test vessels. The surviving P0 generation was transferred to the new solutions, using a wide bore pipette, minimising transfer of medium.
- No. of organisms per vessel: one D. magna, (<24 hours old) - termed the P0 generation
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water medium used for testing (and maintenance of stock cultures) was Elendt's M4 D. magna medium and is described in Appendix 3 (attached). The dilution water was aerated for >2 hours before use.
- The pH, total hardness, conductivity, alkalinity and total chlorine were determined for the batch of dilution water used in the test. Ammoniacal nitrogen as N, suspended solids, total organic carbon (TOC) and chemical oxygen demand (COD) were determined for a recently analysed batch, along with a range of metals, pesticides and PCBs. This non-study background data is shown in Appendix 4 (attached).
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light:8 hours dark, with a 20-minute dawn: dusk transition period
- Light intensity: Light intensity, measured three times during the study initiation at test vessel height, with a mean of 1374 Lux.
- Feeding: The test D. magna were fed daily using a mixed diet of YCT (yeast cereal trout) and Pseudokirchneriella subcapitata, strain CCAP 278/4.
The P. subcapitata cultures were harvested, centrifuged and measured for cell density using a Coulter Counter. The algal cells were then re-suspended in D. magna culture medium, stored in a refrigerator, in the dark and used within two weeks.
YCT was prepared by mixing equal volumes of yeast, cereal and trout food supernatants and stored frozen for a maximum of 3 months, until required. Thawed food is stored in a refrigerator, in the dark and used within two weeks.
The P. subcapitata stock, once re-suspended, and YCT were analysed for total organic carbon content. The feeding was designed so that each test vessel received a nominal 0.06 mg/C/day of algae and 0.06 mg/C/day of YCT suspension, providing a total of 0.12 mg/C/day.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations of survival of the P0 generation were made daily
- Other symptoms of toxicity observed (for example presence of eggs in brood pouch, presence of males or ephippia eggs) were recorded daily
- Observations were made daily for the presence of offspring (termed the F1 generation) in each vessel, if present the number of offspring were counted, recorded and removed.
- At the end of the test the surviving P0 Daphnia were measured. To do this, the excess liquid surrounding the Daphnia was removed and the length (apex of helmet to base of spine) measured using a microscope with an eyepiece graticule previously calibrated with a stage micrometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 2.15

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 0.09 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

body length of P0

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

body length of P0

Details on results:

Survival data
Mortalities of P0 generation D. magna by replicate are given in Table 3. No significant difference in survival was found between the control and all the exposure concentrations.
One mortality was observed in the nominal 0.09 mg a.i./L concentration on day 8 and in one of the replicates of exposure treatment was observed to be paler than the control and 8 aborted eggs were observed in the test vessel. In the nominal 0.43 mg a.i./L concentration, one mortality was observed on day 6. A further two mortalities were observed over days 13 and 14, resulting in 30% mortality in the exposure concentration by study end. One mortality was observed in the nominal concentration 0.90 mg a.i./L on Day 6 followed by one further mortality on Day 13. The nominal concentration of 2.0 mg a.i./L had one mortality on each of days 9 and 19.
The results obtained from the survival analysis, based on nominal concentrations, are as follows:
NOEC ≥ 2.0 mg a.i./L
LOEC > 2.0 mg a.i./L
EC50 > 2.0 mg a.i./L
EC20 = 0.388 mg a.i./L; 95 % confidence limits = 0.286 mg a.i./L - N.D

Reproduction data
The total numbers of offspring (F1) obtained from each surviving P0 D. magna at test end and in the start of the test which did not die accidently or inadvertently during the test are given in Table 4 and Table 5, respectively and are shown in Figure 1 and Figure 2, respectively (attached). Also shown are the arithmetic means, standard deviations, coefficient of variation and totals.
The P0 D. magna in all nominal measured test concentrations released their first offspring between days 7 – 9. In the dilution water control the first offspring were released between days 8 – 9.
The surviving D. magna in the dilution water control and nominal 0.09, 0.20, 0.90 and 2.0 mg a.i./L concentrations had at least five broods by the end of the study. The surviving D. magna in nominal 0.09 mg a.i./L concentration had completed four broods by the end of the study.
Aborted eggs were visible on the bottom of the vessel Day 12 in one replicate of the nominal 0.90 mg a.i. /L concentration.

Number of offspring per surviving P0 D. magna at test end
The mean number of offspring produced, per surviving parent, at the end of the test was 99 in the dilution water control. The residual standard deviation (coefficient of variation) around the mean number of living offspring per parent in the dilution water control was 12%, below the maximum of 25% recommended in the guideline.
The mean number of offspring produced per surviving P0 D. magna at test end in the exposure concentrations ranged from 102 to 132.
No statistically significant difference (reduction) in reproduction (p < 0.05) was found between the control and exposure concentrations.
The results obtained from the reproduction analyses, based on nominal concentrations, are as follows:
reproNOEC ≥ 2.0 mg a.i./L
reproLOEC > 2.0 mg a.i./L
reproECx values not determined

Number of offspring produced per P0 D. magna in the start of the test which did not die accidently or inadvertently during the test
The mean number of offspring produced per P0 D. magna in the start of the test which did not die accidently or inadvertently during the test ranged from 92 to 119.
No statistically significant difference (reduction) in reproduction (p < 0.05) was found between the dilution water control and exposure concentrations.
The results obtained from the reproduction analyses, based on nominal concentrations, are as follows:
reproNOEC ≥ 2.0 mg a.i./L
reproLOEC > 2.0 mg a.i./L
reproECx values not determined

Body length data
The body lengths of the surviving P0 D. magna at the end of the test are given in Table 5 along with the mean and standard deviation.
The results obtained from the length analyses, based on nominal concentrations, are as follows:
lengthNOEC < 0.09 mg a.i./L
lengthLOEC = 0.09 mg a.i./L
lengthEC50 > 2.0 mg a.i./L
lengthEC20 > 2.0 mg a.i./L
A statistically significant difference in length (reduction) was found between the control and all exposure concentrations, however, due to the lack of dose response observed and the ECx values were greater than 2 mg a.i./L the results are not considered statistically or biologically reliable.

Reported statistics and error estimates:

Survival data
Significant differences (p< 0.05) between the control and treatment groups were analysed using a Fisher Exact test with Bonferroni-Holm correction.
The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation (ICPIN) method.
Reproduction data
Number of offspring per surviving P0 D. magna at test end
The control and test concentration data were not normally distributed (Shapiro-Wilk’s test) and data variances were equal (Levene’s test). Significant differences (p < 0.05) between the control and test concentrations were analysed using a Williams Multiple Sequential t-test.
The EC50, EC20, and EC10 values with their associated confidence intervals were subsequently calculated using the Nonlinear Regression (NLR) method.
Number of offspring produced per P0 D. magna in the start of the test which did not die accidently or inadvertently during the test
The control and test concentration data were not normally distributed (Shapiro-Wilk’s test) and data variances were equal (Levene’s test). Significant differences (p < 0.05) between the control and test concentrations were analysed using a Multiple Sequentially- rejective Median Test after Bonferroni- Holm test.The EC50, EC20, and EC10 values with their associated confidence intervals were subsequently calculated using the Nonlinear Regression (NLR) method.
Body length data
The control and test concentration data were not normally distributed (Shapiro-Wilk’s test), and data variances were unequal (Bartlett Equality test). Significant differences (p < 0.05) between the control and test concentrations were analysed using a Jonckheere-Terpstra Step-Down test.
The EC50, EC20, and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation (ICPIN) method.

The statistical analysis reports are attached (Appendix 8)

Analytical data

The measured concentrations of the four major individual components of the test substance (C12, C14, C16 and C18-α-sulfo, 1-methyl esters, sodium salts) in the test solutions and the fortified solutions are given in Table 1 and Table 2, respectively (attached). Typical chromatograms are shown in Appendix 5 (attached), Figures 1-10. Typical calibration curves are shown in Appendix 5, Figure 11. All analytical values are quoted to three significant figures and percentages to the nearest integer.

The individual pooled sample of each test concentration replicate was sampled for each treatment and measured at each time point. The arithmetic mean results for both ‘on’ and ‘off’ solutions are given in Table 1. Analytical procedural recovery samples of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts are given in Table 2. The limit of quantification of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts was determined to be 10 μg/L during the method validation study.

As multiple components of the test substance were measured, nominal concentrations were used for calculation and reporting of results, with measured concentrations for each individual component presented separately.

Measured concentration of monosodium salts

The dilution water control was measured and determined to be <LOQ for all measured components throughout the exposure. Measured concentrations of the four major individual components during the exposure (Table 1) were as follows:

- C12-α-sulfo, 1-methyl esters, monosodium salts ranged between 35-144% of nominal;

- C14-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-126% of nominal;

- C16-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-104% of nominal;

- C18-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-124% of nominal.

Throughout the study the analysis of the monosodium-C12 component was inconsistent, with notable levels of response drift during the analytical sequences. Frequent placement of suitable concentration quality control standards allowed assessment of the level of response drift and subsequent adjustment of the analytical results for the study samples and other unknown solutions in accordance with Scymaris’ standard operating practice. In some instances, the associated acceptance criteria were not fully met. In each case this is clearly identified in the data tables. Therefore, these data should be treated with caution but are included here for consistency.

On Day 13 and Day 14 Mono-C12 failed to meet all critical analytical method validity criteria. The following criteria failed: response drift throughout run. All samples, following response drift were quantified using bracketing standards quantitation which did not fully pass acceptance criteria. The only part of the test sequence where the response drift could not be accounted for was used to analyse the spike samples (and DWC sample), therefore, these results are not reported. All other data passed the acceptance criteria with the use of bracketing standards quantitation.

Measured concentrations of the fortified recovery samples for the four major individual components during the exposure (Table 2) were as follows:

- C12-α-sulfo, 1-methyl esters, monosodium salts ranged between 45-221% of nominal;

- C14-α-sulfo, 1-methyl esters, monosodium salts ranged between 81-104% of nominal;

- C16-α-sulfo, 1-methyl esters, monosodium salts ranged between 82-113% of nominal;

- C18-α-sulfo, 1-methyl esters, monosodium salts ranged between 89-117% of nominal.

Measured concentration of disodium salts

Measured concentrations of the four minor individual components of the test substance (C12, C14, C16 and C18-α-sulfo, disodium salts) in the test solutions and the fortified recovery samples are given in Appendix 6 and Appendix 7 respectively (attached).

Table 3 Survival data

Nominal concentration of Fatty acids, C12-18(even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)

Survival of P0Daphnia magnaat 21 days

1

2

3

4

5

6

7

8

9

10

Total survival

Percentage Survival (%)

Dilution water control

A

A

A

A

A

A

A

A

A

A

10

100

0.09

A

M

A

A

A

A

A

A

A

A

9

90

0.20

A

A

A

A

A

A

A

A

A

A

10

100

0.43

A

M

A

A

A

A

A

A

M

M

7

70

0.90

A

A

A

A

A

A

M

A

M

A

8

80

2.00

A

A

M

A

A

A

M

A

A

A

8

80

A = Alive, M = Mortality

Table 4 Reproduction data per surviving P0D. magna

Nominal concentration of Fatty acids, C12-18(even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Total number of offspring per surviving P0Daphnia magna										Mean per P0 D. magna	Standard deviation	Total offspring	CV	Mean offspring as % of control
	1	2	3	4	5	6	7	8	9	10
Dilution water control	77	108	99	101	112	108	81	95	106	100	99	11.59	987	11.74	-
0.09	121	M	92	117	128	121	108	117	124	106	115	11.1	1034	9.7	116
0.20	97	105	105	97	112	103	106	105	95	94	102	5.8	1019	5.73	103
0.43	124	M	121	123	112	123	123	112	M	M	120	5.3	838	4.5	121
0.90	131	136	128	153	123	113	M	130	M	106	128	14.3	1020	11.23	129
2.00	124	122	M	154	123	124	M	136	137	134	132	10.9	1054	8.3	133

M = Mortality, CV = Coefficient of Variation.

Arithmetic mean used, excluding mortalities.

Table 5 Reproduction data per P0D. magnaat test start which did not die accidently or inadvertently during the test

Nominal concentration of Fatty acids, C12-18(even numbered)-methyl esters, sulfonated, sodium salts (mg a.i./L)	Total number of offspring per P0Daphnia magnaat test start which did not die accidently or inadvertently during the test										Mean per P0 D. magna	Standard deviation	Total offspring	CV	Mean offspring as % of control
	1	2	3	4	5	6	7	8	9	10
Dilution water control	77	108	99	101	112	108	81	95	106	100	99	11.6	987	11.7	-
0.09	121	21	92	117	128	121	108	117	124	106	106	31.5	1055	29.8	107
0.20	97	105	105	97	112	103	106	105	95	94	102	5.8	1019	5.7	103
0.43	124	0	121	123	112	123	123	112	30	54	92	46.3	922	50.2	93
0.90	131	136	128	153	123	113	43	130	0	106	106	47.5	1063	44.7	108
2.00	124	122	106	154	123	124	28	136	137	134	119	34.3	1188	28.9	120

M = mortality, CV = Coefficient of Variation.

Arithmetic mean used.

Table 6 Adult body length measurements

Nominal concentration of Fatty acids, C12-18(even numbered)-methyl esters, sulfonated, sodium salts     (mg a.i./L)	Length at day 21 per surviving P0Daphnia magna(mm)										Mean length ± SD (mm)	Mean as % of control
	1	2	3	4	5	6	7	8	9	10
Dilution water control	4.63	4.81	4.75	5.06	5.00	5.00	4.19	4.38	3.81	4.25	4.59 ± 0.42	-
0.09*	4.31	M	4.31	4.13	4.19	4.13	4.19	4.06	4.06	4.00	4.15 ± 0.11	91
0.20*	4.25	4.06	4.06	4.00	4.13	4.06	3.94	4.00	3.88	3.88	4.03 ± 0.11	88
0.43*	4.31	M	4.19	4.25	4.13	4.19	4.25	4.13	M	M	4.21 ± 0.07	92
0.90*	4.13	4.19	4.19	4.19	4.19	4.13	M	4.19	M	3.94	4.14 ± 0.09	90
2.00*	4.06	4.06	M	4.19	4.06	4.06	M	4.13	4.06	4.00	4.08 ± 0.06	89

M = Mortality, SD = standard deviation. Arithmetic mean used. *Significant differences (p < 0.05) found between control and exposure concentrations.

Validity criteria

The OECD 211 Test Guideline details the following performance criteria for the test validity:

- the mortality of the parent animals (P0 Daphnia) in the control(s) should not be more than 20%

- The mean number of live offspring produced per parent animal surviving at the end of the test in the control(s) is >60.

- No ephippia are produced.

There were no mortalities of P0 D. magna in the dilution water control, the mean number of live offspring produced was 99 in the dilution water control and no ephippia were produced. Therefore, the test met the validity criteria for the test guideline.

Validity criteria fulfilled:

yes

Remarks:

(see any other information on results)

Conclusions:

Taking all biological parameters and assessed endpoints into account, there was no effect observed of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts. The determined NOEC and LOEC of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts was ≥2.0 and >2.0 mg a.i./L, respectively.

Executive summary:

Summary:

Subject: Determination of effects on reproduction to Daphnia magna

Guideline: OECD 211 guideline (adopted 2nd October 2012)

Test species: D. magna (Clone A), < 24 hours old

Source of organisms: Continuous laboratory cultures at Scymaris; parental stock age 12 +/- 1 days old

Test concentrations: Control and nominal concentrations of 0.09, 0.20, 0.43, 0.90 and 2.0 mg a.i./L

Length of test: 21 days, Semi-static (renewal every day)

Nominal test temperature: 20 -24, kept constant within +/- 2 °C

Results based on nominal concentrations of Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts:

Endpoints	No observed effect  concentration (NOEC)	Lowest observed effect concentration (LOEC)	EC 50	EC20	EC10
			mg a.i./L
Survival	≥ 2.0 mg a.i./L	> 2.0 mg a.i./L	> 2.0	0.388 (0.286 - N.D.)	N.D.
Length	< 0.09 mg a.i./L	0.09 mg a.i./L	>2.0	>2.0	N.D.
Reproduction (per surviving P0 D. magna at test end)	≥ 2.0 mg a.i./L	> 2.0 mg a.i./L	N.D.*	N.D.*	N.D.*
Reproduction (per surviving P0 D. magna at test start)	≥ 2.0 mg a.i./L	> 2.0 mg a.i./L	N.D.*	N.D.*	N.D.*

N.D. = Not Determined

95% confidence intervals in parentheses

*The ECx could not be statistically calculated and determined to be greater than 2.0 mg a.i./L

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

25 April 1994 to 16 May 1994

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 202 Daphnia Sp., Acute Immobilisation Test and Reproduction Test

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Water samples of fresh media were taken on Day 0 and samples of expired media were taken on Days 2, 4, 7, 9, 11, 14, 16, 18 and 21 from each surviving test and control vessel.
- Chemical analysis was also carried out on the aqueous stock solutions of 1.0, 10, 100 and 1000 mg/L on days 0, 2, 4, 7, 9, 11, 14, 16 and 18.

Vehicle:

no

Details on test solutions:

TEST MATERIAL PREPARATION
- Test material (1.00 g) was dispersed in water with the aid of ultrasonic disruption and the volume was adjusted to give a 1000 mg/L stock solution.
- Serial dilutions were made from the stock solution to prepare further stock solutions of 100, 10 and 1.0 g/L.
- Aliquots of stock solutions were each separately dispersed in reconstituted water and adjusted to give the test series (50 and 160 mL of 1.0 mg/L; 50 and 160 mL of 10 mg/L; 50 mL of 100 mg/L)

Test organisms (species):

other: Daphnia magna Straus

Details on test organisms:

- Source: Laboratory culture originating from a strain supplied by the Institut National de Recherche Chimique Appliquée (I.R.CH.A.) France.
- Culture: Polypropylene vessels at 21 °C containing 2 L of reconstituted water (see Appendix 5, attached). Cultures were fed daily with a suspension of mixed algae (predominantly Chlorella spp.). Culture conditions ensured that reproduction was by parthenogenesis.
- Selection: Gravid adults were isolated 24 hours prior to initiation of the test. Young daphnids produced overnight were used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Reconstituted water had an approximate theoretical total hardness of 270 mg/L as CaCO3

Test temperature:

21.0 °C

pH:

7.6-7.8

Dissolved oxygen:

89 %

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

- Nominal concentrations of 0.025, 0.080, 0.25, 0.80 and 2.5 mg/L

Details on test conditions:

TEST WATER
- Reconstituted water (see Appendix 5, attached).

EXPOSURE CONDITIONS
- Test vessels: Glass flasks containing 400 mL test solution and covered to reduced evaporation.
- Experimental design: Five test concentrations plus one control (4 replicates of each) and 40 animals per concentration.
- Method of initiation: Daphnia were placed in the test solutions after addition of the test substance.
- Loading: 40 mL test solution per organism.
- Photoperiod: 16 hours light and 8 hours dark.
- Aeration: None (the diluent only was aerated prior to test media preparation).
- Medium renewal: Three times per week (days 2, 4, 7, 9, 11, 14, 16 and 18).

OBSERVATIONS
- Temperature was recorded daily for each flask.
- Dissolved oxygen, pH and temperature were measured before and after each test material renewal.
- Live and dead Daphnia of the parental (P1) generation were counted daily.
- General condition and size of the Daphnia as compared with the control were assessed at each test media renewal period.
- The number of Daphnia with eggs or young in the brrod pouch was determined at each test media renewal together with the numbers of live and dead filial (F1) Daphnia.
- The number of discarded unhatched eggs was also determined at each test media renewal.

METHOD OF RENEWAL
- Adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel was passed through a fine mesh.
- Young daphnids (live and dead) and unhatched eggs collected on the mesh were counted using a stereo microscope and then discarded.
- Young daphnids were considered to be dead if no sign of movement was apparent duric microscopic examination.
- Adult Daphnia that were unable to swim for approximately 15 seconds after gentle agitation were considered to be dead.
- An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of offspring produced in the flasks.

FEEDING
- Each vessle received approximately 10 μL of a mixed unicellular algal culture (equivalent to approximately 4.2 x 10E09 cells/mL) daily.
- Feeding was at a level to maintain a green tinge in the test solutions thereby ensuring food was available continuously.
- Equal amounts of food were given to each daphnid (equivalent to approximately 0.28 mg C/daphnid/day) throughout the duration of the study.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % confidence limits

Remarks:

0.44 to 0.53 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

>= 0.25 - <= 0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation and reproduction

Details on results:

RESULTS
- Observations are summarised in Tables 1 to 6 (attached).
- Total cumulative production of young is given in Table 7 (attached).
- Number of young produced per adult is shown in Table 8 (attached).
- Cumulative production of young per adult is presented in Figure 1 (attached).
- Environmental measurements (pH, temperature and oxygen concentration) are summarised in Appendix 1 (attached).
- Data for individual replicates are given in Appendix 3 (attached).

LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- Mortality (immobilisation) occurred predominantly within the first 48 hours of exposure at the highest test concentration of 2.5 mg/L resulting in 40 % mortality after 48 hours, 63 % mortality by 7 days and 100 % mortality by 11 days.
- Significant mortality (immobilisation) indicating a prolonged toxic effect attributable to exposure of Daphnia magna to test material also occurred throughout the study in the 0.80 mg/L test group resulting in 13 % mortality by 14 days and 93 % mortality by 21 days.
- No significant mortalities occurred at test concentrations of 0.025, 0.080 and 0.25 mg/L throughout the duration of the study.
- EC50 (immobilisation) values based on nominal test concentrations (see table, below) were calculated using the method of Thompson (1947).
- It was not possible to calculate the 48-hour EC50 value as < 50 % mortalities were observed at that time point. However, a mortality rate of > 50 % was observed after 72 hours enabling calculation of an EC50 value.
- There was no significant effect on size of daphnids as a result of exposure to test material. However, Daphnia were observed to be markedly paler in colour than the control animals. This effect was seen at 2.5 mg/L on day 7 (4/15 daphnids) and day 9 (10/10 daphnids); 0.80 mg/L on day 11 (12/37 daphnids), day 14 (21/35 daphnids), day 16 (15/25 daphnids), day 18 (14/16 daphnids) and day 21 (3/3 daphnids).

SUB-LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- There were no statistically significant differences in terms of the number of young produced per adult between controls and the 0.025, 0.080 and 0.25 mg/L test groups (P ≥ 0.05) after both 14 and 21 days.
- The 0.080 mg/L test group showed a statistically significant difference from the controls and the 0.025, 0.080 and 0.025 mg/L test groups on day 14 in terms of the numbers of young produced per adult.
- The 2.5 mg/L test group produced no young due to the acute effects of exposure to the test material.
- The EC50 (reproduction) after 14 and 21 days could only be estimated given the unsuitable nature of the data for standard calculation of EC50 values.
- The estimated EC50 (reproduction) for 14 and 21 days exposure was considered to be between 0.25 mg/L (4% fewer young per adult than the control group over 21 days) and 0.80 mg/L (58 % fewer young per adult than the control group over 21 days).
- The 0.25 mg/L test group produced approximately 96 % of the total number of young produced by the control group.
- The 0.80 mg/L test group produced approximately 33 % of the total number of young produced by the control group.

EFFECTS ON THE FILIAL GENERATION (F1)
- Information on the effects of test item on the F1 generation was limited because the study design meant young were removed soon after liberation from the brood pouch.
- Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

NO OBSERVED EFFECT CONCENTRATION (NOEC)
- The NOEC was given as 0.25 mg/L because at this concentration there were no significant differences (P ≥ 0.05) in terms of the numbers of young produced per adult compared to controls and, at this test concentration, there were no mortalities (immobilisation) observed in the parental generation (P1).

VERIFICATION OF TEST CONCENTRATIONS
- Care should be taken in the interpretation of the test results due to apparent presence of test material in the control and high measured concentrations for the lowest test concentration of 0.025 mg/L.
- The method of analysis was not specific to the test material and it was considered that residual detergent in the test/analytical apparatus was responsible for the elevated concentrations in the control and the and the lowest test concentration.
- Chemical analysis of freshly prepared test media on day 0 (see Appendix 2, attached) showed the measured test concentrations to range between 85 and 200 % of nominal.
- Analysis of the expired test media on days 2, 4, 7, 9, 11, 14, 16, 18 and 21 (see Appendix 2, attached) showed measured test concentrations ranging between 9.3 to 408 % of nominal.
- The higher percentage measured concentrations were observed in the lower test concentrations and were considered to be due to residual detergent from the test/analytical glassware.
- Measured test concentrations for the 0.080, 0.25 and 0.80 mg/L test groups were observed to decline after day 11 of the study. This effect was considered to be due to a build-up of bacteria, introduced with the algal cells that were used to feed the Daphnia, resulting in a gradual biological degradation of the test material.
- Chemical analysis of the aqueous stock solutions used to prepare the test series on days 0, 2, 4, 7, 9, 11, 14, 16 and 18 showed the measured concentrations to be near nominal on all sampling occasions (see Appendix 2, attached).

Reported statistics and error estimates:

- Details of statistical analysis are given in Appendix 4 (attached).

Validation criteria	Required	Actual
Control mortality	≤ 20 %	0 %
Dissolved oxygen	≥ 60 %	89%
pH (controls)	Deviation ≤ 0.3	0.1
First young (control group)	Produced within 9 days	8 days
Cumulative young per female (control group)	≥ 20 after 14 days ≥ 40 after 21 days	22 48
Number of broods per control group	≥ 3	6-7*
* Pooling of data from the four replicates tends to mask identification of distinct broods

Time	EC50 (mg/L)	95 % confidence limits (mg/L)
24 h	> 2.5	-
48 h	> 2.5	-
72 h	2.4	1.7-3.3
7 d	2.0	1.6-2.5
14 d	1.2	1.1-1.4
21 d	0.48	0.44-0.53

Validity criteria fulfilled:

no

Conclusions:

Exposure of Daphnia magna to test material resulted in an immediate lethal effect at a concentration of 2.5 mg/L. A significant prolonged mortality effect occurred in the adult Daphnia and significant impairment of reproduction was observed at a test item concentration of 0.80 mg/L. Results were reported as EC50 (21 d) 0.48 mg/L based on immobilisation and EC50 (21 d) 0.25-0.80 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was given as 0.25 mg/L based on immobilisation and reproduction.

Executive summary:

The effect of test item on reproduction of Daphnia magna was assessed using the acute immobilisation test and reproduction test (OECD 202; 1984 version). Daphnia magna were exposed in four replicates to five nominal concentrations of test material plus reconstituted water as the control for 21 days under semi-static conditions (40 animals per concentration; 10 daphnids per replicate). Nominal concentrations of test item were 0.025, 0.080, 0.25, 0.80 and 2.5 mg/L. Verification of the control and test item concentrations took place on days 2, 4, 7, 9, 11, 14, 16, 18 and 21. Chemical analysis was also carried out on the aqueous stock solutions of 1.0, 10, 100 and 1000 mg/L on days 0, 2, 4, 7, 9, 11, 14, 16 and 18. Measured concentrations ranged from 9.3 % to 408 % of nominal with the higher percentage concentrations being observed in the lower test concentrations. Since the analytical method was not specific to the test material, this effect was considered to be caused by residual detergent on the test/analytical glassware. Analysis of the aqueous stock solutions used to prepare the test series showed measured concentrations to be near nominal on all occasions. Exposure of Daphnia magna to test material resulted in an immediate lethal effect at a concentration of 2.5 mg/L. A significant prolonged mortality effect occurred in the adult Daphnia and significant impairment of reproduction was observed at a test item concentration of 0.80 mg/L (the 0.25 mg/L test group produced 4 % fewer young than the control and the 0.80 mg/L test group produced 58 % fewer young than the control group). Results were reported as EC50 (21 d) 0.48 mg/L based on immobilisation and EC50 (21 d) 0.25-0.80 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was given as 0.25 mg/L based on immobilisation and reproduction.

Description of key information

Taking all biological parameters and assessed endpoints into account, the determined 21-d NOEC and LOEC was ≥2.0 and >2.0 mg a.i./L, respectively (Daphnia magna; OECD TG 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 2 mg/L

Additional information

The chronic toxicity of the registered substance to D. magna was determined in a GLP study performed according to OECD TG 211, Daphnia magna Reproduction Test. 

D. magna (Clone A), < 24 hours old were exposed to the registered substance for 21 days at nominal concentrations of 0 (control) 0.09, 0.20, 0.43, 0.90 and 2.0 mg a.i./L. A semi-static (renewal every day) test design was used with ten replicates per treatment. The nominal test temperature was 20 -24, kept constant within ± 2 °C. 

To establish the concentrations achieved eight components of the registered substance in the test solutions were measured using a liquid chromatography – mass spectrometry (LC-MS/MS) method. The analytical method was successfully validated for the analysis of three of the major individual components of the test substance (C12, C14 and C18-α-sulfo, 1-methyl esters, sodium salts). The method also measured C16-α-sulfo, 1 methyl esters, sodium salts (which did not meet the acceptance criteria of the method validation study) and four of the minor individual components of the test substance (C12, C14, C16 and C18-α-sulfo, disodium salts) but measurement of these components using this method was not validated. 

The individual pooled sample of each test concentration replicate was sampled for each treatment and measured at each time point.

Measured concentrations of the four major individual components during the exposure were as follows:

- C12-α-sulfo, 1-methyl esters, monosodium salts ranged between 35-144% of nominal;

- C14-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-126% of nominal;

- C16-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-104% of nominal;

- C18-α-sulfo, 1-methyl esters, monosodium salts ranged between <LOQ-124% of nominal.

Throughout the study the analysis of the monosodium-C12component was inconsistent, with notable levels of response drift during the analytical sequences. Frequent placement of suitable concentration quality control standards allowed assessment of the level of response drift and subsequent adjustment of the analytical results for the study samples and other unknown solutions in accordance with Scymaris’ standard operating practice. In some instances, the associated acceptance criteria were not fully met. Therefore, these data should be treated with caution but are included for consistency.

On Day 13 and Day 14 Mono-C12 failed to meet all critical analytical method validity criteria. The following criteria failed: response drift throughout run. All samples, following response drift were quantified using bracketing standards quantitation which did not fully pass acceptance criteria. The only part of the test sequence where the response drift could not be accounted for was used to analyse the spike samples (and DWC sample), therefore, these results are not reported. All other data passed the acceptance criteria with the use of bracketing standards quantitation.

Measured concentrations of the fortified recovery samples for the four major individual components during the exposure were as follows:

- C12-α-sulfo, 1-methyl esters, monosodium salts ranged between 45-221% of nominal;

- C14-α-sulfo, 1-methyl esters, monosodium salts ranged between 81-104% of nominal;

- C16-α-sulfo, 1-methyl esters, monosodium salts ranged between 82-113% of nominal;

- C18-α-sulfo, 1-methyl esters, monosodium salts ranged between 89-117% of nominal. 

As multiple components of the test substance were measured, nominal concentrations were used for calculation and reporting of results, with measured concentrations for each individual component presented separately.

No significant difference in survival of P0 generation D. magna was found between the control and all the exposure concentrations. One mortality was observed in the nominal 0.09 mg a.i./L concentration on day 8 and in one of the replicates of exposure treatment was observed to be paler than the control and 8 aborted eggs were observed in the test vessel. In the nominal 0.43 mg a.i./L concentration, one mortality was observed on day 6. A further two mortalities were observed over days 13 and 14, resulting in 30% mortality in the exposure concentration by study end. One mortality was observed in the nominal concentration 0.90 mg a.i./L on Day 6 followed by one further mortality on Day 13. The nominal concentration of 2.0 mg a.i./L had one mortality on each of days 9 and 19.

The results obtained from the survival analysis, based on nominal concentrations, are as follows:

NOEC ≥ 2.0 mg a.i./L; LOEC > 2.0 mg a.i./L; EC50 > 2.0 mg a.i./L; EC20 = 0.388 mg a.i./L; 95 % confidence limits = 0.286 mg a.i./L - N.D

The P0 D. magna in all nominal measured test concentrations released their first offspring between days 7 – 9. In the dilution water control the first offspring were released between days 8 – 9. The surviving D. magna in the dilution water control and nominal 0.09, 0.20, 0.90 and 2.0 mg a.i./L concentrations had at least five broods by the end of the study. The surviving D. magna in nominal 0.09 mg a.i./L concentration had completed four broods by the end of the study. Aborted eggs were visible on the bottom of the vessel Day 12 in one replicate of the nominal 0.90 mg a.i. /L concentration.

The mean number of offspring produced per surviving P0 D. magna at test end in the exposure concentrations ranged from 102 to 132. No statistically significant difference (reduction) in reproduction (p < 0.05) was found between the control and exposure concentrations.

The results obtained from the reproduction analyses, based on nominal concentrations, are as follows:

NOEC ≥ 2.0 mg a.i./L; LOEC > 2.0 mg a.i./L; ECx values not determined

The mean number of offspring produced per P0 D. magna in the start of the test which did not die

accidently or inadvertently during the test ranged from 92 to 119. No statistically significant difference (reduction) in reproduction (p < 0.05) was found between the dilution water control and exposure concentrations.

The results obtained from the reproduction analyses, based on nominal concentrations, are as

follows:

NOEC ≥ 2.0 mg a.i./L; LOEC > 2.0 mg a.i./L; ECx values not determined

The results obtained from the length analyses, based on nominal concentrations, are as follows:

NOEC < 0.09 mg a.i./L; LOEC = 0.09 mg a.i./L; EC50 > 2.0 mg a.i./L; EC20 > 2.0 mg a.i./L

A statistically significant difference in length (reduction) was found between the control and all exposure concentrations, however, due to the lack of dose response observed and the ECx values

were greater than 2 mg a.i./L the results are not considered statistically or biologically reliable.

Taking all biological parameters and assessed endpoints into account, there was no effect observed of the registered substance. The determined 21-d NOEC and LOEC was ≥ 2.0 and > 2.0 mg a.i./L, respectively.