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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-07 to 2019-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch number: 1802000003
CAS No.: 186817-80-1
Purity (certified): 98 %
Analytical monitoring:
yes
Details on sampling:
- Concentrations:0 - 0.316 - 1.00 - 3.16 - 10.0 - 31.6 mg/L (nominal)
- Sampling method: All concentration levels and the control were analysed via GC-MS 3 times during the test (e.g. once within a period of 7 days) in the fresh media at the start of an exposure interval (0 hours) and in the old media at the end of an exposure interval (24 hours).
At the start of the exposure intervals, samples of the fresh media were taken after preparation of all test item concentrations and analysed. At the end of the exposure intervals (24 hours), samples of the old media were taken directly from the test vessels.
- Sample storage conditions before analysis: All samples were immediately extracted, and the extracts were stored at room temperature if necessary. Until the start of the analysis the prepared samples were stored on an autosampler.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 31.6 mg/L of the test item was freshly prepared with test medium for each exposure interval. An appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of the test medium. The stock solution was stirred for 10 minutes at room temperature and 1100 rpm. The stock solution was used as the highest concentration level and for the preparation of further test concentrations by dilution with the test medium.
Five concentrations of the test item were prepared by dilution of the stock solution of 31.6 mg/L with the test medium and tested at follows:
0.316 - 1.00 - 3.16 - 10.0 - 31.6 mg/L (separation factor √10),
which corresponds to the time-weighted mean measured test item concentrations of:
0.234 - 0.765 – 2.40- 7.56 – 24.6 mg/L
- Differential loading: n.a.
- Controls: 10 replicates of dilution water without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle applied.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No.
- Other relevant information: /
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Not applicable
- Justification for species other than prescribed by test guideline: n.a.
- Age at study initiation (mean and range, SD): < 24 h
- Stage and instar at study initiation: first instar;
- Method of breeding: In glass vessels (2–3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, 16 hours illumination; light intensity of max. 1500 lx
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany; Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Feeding during test: Yes
- Food type and amount: Pseudokirchneriella subcapitata (0.386–0.977 mL) and Desmodesmus subspicatus (0.213–0.480 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. The density of the algae suspension varied, but was the same for all test groups on each feeding day.
- Frequency: Daily

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS:
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
260 to 277 mg CaCO3/L
Test temperature:
19.0 to 21.0 °C
pH:
7.31 to 7.88
Dissolved oxygen:
6.84 to 10.45 mg dissolved O2 /L
Nominal and measured concentrations:
nominal: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 mg/L
measured (time-weighted mean): 0.234 - 0.765 – 2.40- 7.56 – 24.6 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5 (ID) × 8 (H) cm), 100 mL capacity, loosely covered with watch glasses
- Test volume: 50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): daily (For this purpose, a second test solutions set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Type: Elendt M4 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in fresh media at the start (0 hours) and in old media at the end (24 hours) of an exposure-renewal interval, in one replicate of the control and the highest test concentration. The water quality parameters in fresh media at the start of an exposure-renewal interval were measured in an additional replicate without daphnids of the highest test concentration and the control. At the end of an exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest test concentration and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test period with a thermo-hygrograph.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of parental daphnids: The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the offspring was counted. Dead specimens were removed as soon as they were detected.
- Neonates: First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of observation were recorded.
- Additional observations/ Condition of parental daphnids: Abnormalities (e.g. abnormal swimming behaviour, number of males and winter eggs) were observed and recorded on each day of observation.
- Total body length / Mean dry weight of parental daphnids: At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all test concentrations and the control were determined. As stated in the OECD protocol, this may be useful additional information but is not used for determination of a NOEC.
- Additional endpoints: The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10

RANGE FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
- Conditions: A non-GLP preliminary range finding test was conducted at the test facility as a shortened reproduction test (until observation of the first brood in all test groups) under semi-static conditions with renewal of the test solutions three times per week over a period of 14 days. Five daphnids, one daphnia per replicate, were exposed to each concentration level and the control.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.52 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
4.22 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
7.56 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Behavioural abnormalities: not reported
- Observations on body length and weight: The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.234 to 24.6 mg/L were in the range of 2.86 to 4.10 mm per daphnid and 4.00 mm per daphnid in the control group. The mean dry weight of the survived parental daphnids was in the range of 0.06 to 0.43 mg per daphnid in the concentration levels 0.234 to 24.6 mg/L and 0.42 mg per daphnid in the control.
- Other biological observations: See below, "Any other information on results including tables".
- Mortality of control: 10 %
- Abnormal responses: No males or ephippia were observed in the control or in the test groups during the test. In the concentration levels 0.765 to 24.6 mg/L, stillborn juveniles or aborted eggs were observed during the exposure period of 21 days. For details see below, "Any other information on results including tables".
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
The intrinsic rate of natural increase (IR) was calculated for the survived parental daphnids with the following EULER-LOTKA-equation with ToxRat Professional.
The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it is zero. For growing populations, it is positive and for shrinking populations, it is negative. Clearly, the latter is not sustainable and ultimately leads to extinction.
Software for statistical evaluation and determination of effect concentrations: All data are computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- Excel, MICROSOFT CORPORATION
- ToxRat Professional, TOXRAT SOLUTIONS GMBH

Reproductive output

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 68.8 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

One parental daphnid, concentration level 7.56 mg/L replicate 2, was excluded from the evaluation of the reproductive output due to accidental mortality by physical injury of the daphnid during transfer. Since the Cochran- Armitage test procedure revealed no significant trend (p ≤ 0.05) in adult mortality (i.e. the occurred mortality did not follow a dose-response pattern), the evaluation was done based on the cumulative offspring per survived parent and those replicates with inadvertent parental mortality were excluded. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels 7.56 and 24.6 mg/L (Multiple sequentially-rejective Welch-t-test after Bonferroni-Holm, α = 0.05). The determination of the EC10- and the EC50-value and their 95 % confidence limits was carried out by probit analysis. The EC10 (reproduction) for the test item 2-ethylhexyl-S-lactate was 2.52 mg/L (confidence limits: 2.02–2.96 mg/L). The EC50 (reproduction) was 4.22 mg/L (confidence limits: 3.65–4.92). The LOEC(reproduction was 7.56 mg/L. The NOEC (reproduction) was 2.40 mg/L. For details seeTable 1.

Table1: Effects on Reproduction for all Surviving Parents (based on the time-weighted mean measured concentrations of the test item)

Time-weighted mean measured

test item concentrations

[mg/L]

Mean number of living offspring
per survived parental daphnid (cumulative value)

Mean

SD

CV

24.6

0

-

-

7.56

0.6

1.13

198

2.40

80.5

11.0

13.7

0.765

74.7

9.09

12.2

0.234

71.1

9.30

13.1

Control

68.8

8.14

11.8

 

Adult Mortality

Accidental mortality as defined in the OECD test guideline was observed at the concentration level 7.56 mg/L (replicate 2) by physical injury of the daphnid during transfer. The test item induced no statistically significant adult mortality after 21 days. The Cochran-Armitage test procedure revealed no significant trend (α = 0.05) in adult mortality.The Cochran-Armitage test confirmed that the occurred mortality did not follow a concentration-response pattern. Those replicates with parental mortality were excluded from the analysis of the test result, as the mortality is unlikely to be substance-related (inadvertent mortality). For details see Table 2.

 

Table2: Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

Time-weighted mean measured

test item concentrations

[mg/L]

Adult Mortality [%]

7 days

14 days

21 days

24.6

0

0

30

7.56*

0

0

22

2.40

0

0

0

0.765

0

20

40

0.234

0

20

20

Control

0

10

10

* One parent daphnia died accidentally and is excluded from the evaluation; replicate number is 9 for this concentration.

 

Intrinsic Rates of Natural Increase (IR)

The intrinsic rates of natural increase (IR) of the survived parental daphnids accounting for generation time and number of living offspring were used for calculation of population growth and maintenance. For details see Table 3.

Table 3: Intrinsic Rates of Natural Increase

Time-weighted mean measured

test item concentrations

[mg/L]

Mean IR

CV

MV±SD

[%]

24.6

--

7.56

-0.056±0.2496

446

2.40

0.347±0.0161

4.6

0.765

0.333±0.0177

5.3

0.234

0.319±0.0260

8.1

Control

0.324±0.0276

8.5

--  = not applicable, due to the mortality of the parental daphnid(s)

MV = mean value

CV = Coefficient of variation

 

First Appearance of living Juveniles

The first appearance ofliving juveniles was observed between days 8 and 10 with the introduced parental daphnids of the control and the concentration levels 0.234 to 2.40 mg/L. In the concentration level 7.56 mg/L only on day 14 living juveniles were observed in two replicates. No living juveniles occurred in the concentration level 24.6 mg/L. However, some daphnids died before producing juveniles or producing only dead juveniles. For details see Table 4.

 

Table 4: First Appearance of Living Juveniles in the Individual Groups

Time-weighted mean measured test item concentrations

[mg/L]

Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.

First

appearance

1

2

3

4

5

6

7

8

9

10

mean day

24.6

--

--

--

--

--

--

--

--

--

--

--

7.56

14

--

--

--

--

14

--

--

--

--

14.0

2.40

8

8

8

8

8

8

8

8

8

8

8.0

0.765

8

8

8

8

8

8

8

8

8

8

8.0

0.234

8

10

8

8

8

8

8

8

8

8

8.2

Control

8

9

8

8

8

8

8

9

8

8

8.2

--  = not applicable, since no living juveniles occurred

 

 

Stillborn Juveniles and Aborted Eggs

In the concentration levels 0.765 to 24.6 mg/L,stillborn juvenilesoraborted eggs were observedduring the exposure period of 21 days.For details see Table 5 and 6.

 

Table5: Stillborn Juveniles and Aborted Eggs produced by the Introduced Parental Daphnids after 21 Days

Time-weighted mean measured

test item concentrations

[mg/L]

Number of

Total no.

 

å

Number of
introduced adults

n

stillborn

juveniles

aborted

eggs

24.6

0

1

1

10

7.56

3

10

13

9

2.40

1

1

2

10

0.765

2

3

5

10

0.234

0

0

0

10

Control

0

0

0

10

n  = number of the introduced parental daphnids

 


Table 6: Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles per Introduced Parental Daphnids

Time-weighted mean measured

test item concentrations

[mg/L]

Number of juveniles

Percentage of

dead juveniles#)

[%]

Dead

Alive

Total

24.6

1

0

1

100.0

7.56

13

3

16

81.3

2.40

2

805

807

0.2

0.765

5

572

577

0.9

0.234

0

590

590

0.0

Control

0

625

625

0.0

Dead = aborted eggs + stillborn juveniles

Total = dead + alive juveniles

#)       = related to the total number of juveniles

 

 

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.234 to 24.6 mg/L were in the range of 2.86 to 4.10 mm per daphnid and 4.00 mm per daphnid in the control group.

The mean dry weight of the survived parental daphnids was in the range of 0.06 to 0.43 mg per daphnid in the concentration levels 0.234 to 24.6 mg/L and 0.42 mg per daphnid in the control. For details see Table 7.

 

Table 7: Total Body Length and Dry Weight of the Survived Parental Daphnids

Time-weighted mean measured test item concentrations

[mg/L]

Total length of the survived parent animals [mm]

MV

n

Dry weight

[mg]

Replicate no.

 

 

1

2

3

4

5

6

7

8

9

10

[mm]

 

S

MV

24.6

2.5

--

2.5

3.0

3.0

3.0

3.0

--

3.0

--

2.86

7

0.4

0.06

7.56

4.0

--

--

--

4.0

4.0

3.5

4.0

3.5

4.0

3.86

7

1.0

0.14

2.40

4.0

4.0

4.0

4.0

4.5

4.0

4.0

4.0

4.0

4.5

4.10

10

3.2

0.32

0.765

4.0

4.0

--

4.0

--

4.0

--

4.0

4.0

--

4.00

6

2.6

0.43

0.234

3.5

4.0

--

4.0

4.0

4.5

--

4.0

4.0

4.0

4.00

8

2.7

0.33

Control

4.0

3.5

4.0

4.0

4.0

4.5

4.0

4.0

--

4.0

4.00

9

3.8

0.42

= number of the survived parental daphnids

--   = not applicable, due to the mortality of the parental daphnid(s)

MV = mean value

 

 

Measured Exposure Concentrations in the Test Vessels during the Definitive Test

The test item concentrations of 2-ethylhexyl-S-lactate were analytically verified via GC-MS in fresh media at the start of the exposure intervals on days 0, 7, 14 (0 hours) and in old media at the end of the exposure intervals on days 1, 8, 15 (24 hours) in all concentration levels and the control.

The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 76 to 109 % of the nominal concentrations. At the end of the respective exposure intervals, the measured test item concentrations in the old media were in the range of 42 to 78% of the nominal concentrations.The analytical results are presented in Table 8 and 9.

Since the measured test item concentrations were not within ± 20 % of the nominal concentrations, the time-weighted mean measured concentrations were calculated and used for evaluation of the effect-concentrations. The time-weighted mean measured concentrations are: 0.234 - 0.765 – 2.40- 7.56 – 24.6 mg/L.

Table 8: Measured Concentrations of the Test Item during the Definitive Test (Days 0, 1, 7 and 8)

Sampling date

Day 0

Fresh medium

Day 1

Old medium

Day 7

Fresh medium

Day 8

Old medium

Nominal

test item

concentration

[mg/L]

2-Ethylhexyl-S-Lactate

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

31.6

30.3

96

24.5

78

28.2

89

19.6

62

10.0

9.92

99

6.05

60

9.56

96

5.56

56

3.16

3.08

97

2.09

66

3.03

96

1.67

53

1.00

0.996

100

0.715

72

0.914

91

0.556

56

 0.316

0.316

101

0.210

66

0.345

109

0.134

42

Control

< LOQ

< LOQ

< LOQ

< LOQ

Meas. conc.= measured concentration of the test item, enrichment and dilution factors taken into account

%= percent of the nominal concentration of the test item

LOQ = limit of quantification (0.010 mg/L of the test item)

Table 9: Measured Concentrations and Time-Weighted Mean Measured Concentration of the Test Item during the Definitive Test (Days 14 and 15)

Sampling date

Day 14

Fresh medium

Day 15

Old medium

 

Nominal

test item

concentration

[mg/L]

2 -ethylhexyl-S-lactate

Time-weighted mean

measured test item

concentrations

[mg/L]

Meas.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

%

31.6

26.4

83

19.4

61

24.6

10.0

8.51

85

6.50

65

7.56

3.16

2.79

88

1.99

63

2.40

1.00

0.798

80

0.658

66

0.765

 0.316

0.240

76

0.203

64

0.234

Control

< LOQ

< LOQ

< LOQ

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

% = percent of the nominal concentration of the test item

LOQ = limit of quantification (0.010 mg/L of the test item)

Table 10: Effect concentrations of 2 -ethylhexyl-S-lactate on the reproduction of Daphnia magna (endpoint: cumulative number of living offspring per survived parent daphnia, based on the time-weighted mean measured test item concentrations)

Effect values

2 -ethylhexyl-S-lactate

Time-weighted mean measured
test item concentrations
[mg/L]

EC10 Reproduction

(with 95% confidence limits)

2.52 (95 % Cl: 2.02–2.96)

EC50 Reproduction

(with 95% confidence limits

4.22 (95 % Cl: 3.65 – 4.92)

LOECReproduction

7.56

NOECReproduction

2.40

 

Validity criteria fulfilled:
yes
Conclusions:
The 21-d EC10 of 2-ethylhexyl-S-lactate in Daphnia magna was determined to be 2.52 (95% Cl: 2.02 – 2.96) mg/L (Time Weighted Mean of measured concentrations, TWM).
Executive summary:

The 21-day reproductive toxicity of 2-ethylhexyl-S-lactate to Daphnia magna was studied under semi-static conditions (daily renewal of test solution). Test organisms were exposed to control, and test chemical at measured concentrations of 0.234 - 0.765 – 2.40- 7.56 – 24.6 mg/L (time weighted mean of measured concentrations, TWM). Mortality did not follow a dose-response relation and was therefore not statistically evaluated. The 21-day EC10 based on reproduction was 2.52 (95 % CI: 2.02–2.96). The 21-day NOEC based on reproduction was 2.40 mg/L. The sublethal effects observed but not analysed statistically were intrinsic rate of population increase (IR), first appearance of living juveniles, stillborn juveniles and aborted eggs, growth, presence of males and occurrence of ephippia. Production of offspring in the treated groups indicated that 2-ethylhexyl-S-lactate had an effect on the reproduction at concentrations greater than 2.40 mg/L. Concentrations were determined analytically via GC-MS.

This study is classified as acceptable and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Results synopsis

Test organism age: 1st instar (< 24 h)

Test type: Semi-static

21-d EC10 (reproduction) = 2.52 (95 % Cl: 2.02–2.96) mg/L TWM

21-d EC50 (reproduction) = 4.22 (95 % Cl: 3.65–4.92) mg/L TWM

21-d LOEC (reproduction) = 7.56 mg/L TWM

21-d NOEC (reproduction) = 2.40 mg/L TWM

Description of key information

The 21-d EC10 of 2-ethylhexyl-S-lactate in Daphnia magna was determined to be 2.52 (95% Cl: 2.02–2.96) mg/L (time weighted mean of measured concentrations, TWM). The test was conducted under GLP and according to OECD TG 211.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
2.52 mg/L

Additional information