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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 31, 2017 to November 03, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
High-performance liquid chromatography method
Details on sampling:
At the start of the test additional sacrificial vessels with dispensed test solution were taken for whole sample analysis and at the end of the test, replicate vessels of the dilution water control and each test concentration was taken for whole sample analysis. All test solutions were diluted x2 with THF with the exception of the nominal 10.7 m/L test solution which was diluted by x 4.
Vehicle:
yes
Remarks:
Elendt's M4 D. magna medium
Details on test solutions:
Preparation of test solutions
This study was run with a dilution water control and nominal exposure concentrations of 0.010, 0.032, 0.102, 0.328, 1.05, 3.4 and 10.7 mg/L. A wider spacing of 3.2 was used for this study based on the non-GLP range finding study, which had wide dose response. This ensured a higher likelihood of capturing the desired endpoints in one test. A primary stock concentrate of Test substance, with a nominal concentration of 25 mg/L, was prepared by adding a nominal 0.025 g of test substance to 1000 mL of culture medium. The stock solution was stirred for 1 h and placed in an ultrasonic bath for 10 minutes. The resultant stock was observed to be a colourless, hazy solution and was used to prepare the test solutions. This was achieved by the direct addition of the appropriate amount of concentrate to dilution water in a volumetric flask. The control consisted of culture medium only. The control and nominal 0.010, 0.032 and 0.102 mg/L test solution were clear and colourless. The 0.328 and 1.05 mg/L test solutions were colourless homogeneous solutions with very fine particles present. The 3.4 and 10.7 mg/L test solutions were colourless and hazy homogeneous solutions with very fine particles present. The nominal 0.328, 1.05, 3.4 and 10.7 mg/L test solutions are therefore classified as homogeneous dispersions.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organism
The test organism was the freshwater crustacean, Daphnia magna, obtained from continuous laboratory cultures held at Scymaris. The stock cultures of D. magna were maintained in a reconstituted water medium, the same as the test dilution water, at a temperature of 20 ± 2ºC. The cultures were maintained in 2 L glass vessels with a working volume of 1.6 L. A photoperiod of 16 h’ light: 8 h dark, with 20-minute transition periods was provided. The D. magna cultures were fed on a mixed algae diet of Chlorella vulgaris, strain CCAP 211/12 and Pseudokirchneriella subcapitata, strain CCAP 278/4. The D. magna cultures were fed daily ad libitum depending on age and density of the culture. Culture conditions were such that the D. magna reproduction was by diploid parthenogenesis. D. magna <24 h old, obtained from a single culture vessel, were used for testing. The parent animals were 21 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium since birth. The test organisms and the cultures from which they were obtained showed no evidence of disease before the test period.
Test type:
static
Water media type:
other: Elendt's M4 D. magna medium
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 2ºC
pH:
7.85 to 8.06
Dissolved oxygen:
8.75 to 9.31 mg/L
Nominal and measured concentrations:
0, 0.010, 0.032, 0.102, 0.328, 1.05, 3.4 and 10.7 mg/L (nominal)
0, 0 (0.010), 0 (0.032), 0.12, 0.36, 1.1, 2.8 and 9.3 mg/L (mean measured)
A 10.7 mg/L test concentration was selected as the highest concentration based on the toxicity and dispersibility in a non-GLP range-finding study. A wider spacing of 3.2 was chosen for the range based on the results in the non-GLP range-finding.
Details on test conditions:
Test apparatus
Glass beakers of 250 mL nominal capacity were used as test vessels, with four replicates per test concentration. Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm. The beakers were covered with loose fitting glass lids. The positions of the treatments were randomly allocated within the test area.
Reference substance (positive control):
not specified
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
ca. 2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits (2.0-3.0)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 2.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits (1.7-3.1)
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
ca. 2.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Details on results:
Analytical data
The limit of quantification of test substance in this study was 0.1 mg/Lfor all test solutions with the exception of the nominal 10.7 mg/L which was 0.2 mg/L. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 0-119% of nominal and at the end were 0-133% of nominal based on whole vessel extraction. The nominal 0.010 and 0.032 mg/L test solutions were below the LOQ and as such was not detected at either time point. These concentrations were below the NOEC and as such not having a measured analytical valued does not impact the result of the study. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 30% and less than 20% at levels greater than the LOQ. On the basis of theanalytical data the mean measured concentrations were used for the calculation and reporting of results.

Results

Non-GLP Range-finding Test Summary

A non-GLP range-finding test was conducted prior to study initiation. It was run with a dilution water control and nominal test concentrations of 0.01, 0.1, 1.0, 10 and 100 mg/L. No analytical confirmation of the test concentrations was performed. The range-finding test was performed under the same conditions and in a similar manner as the study with the exception that only two replicates were set up for each test concentration.

Nominal concentration

(mg/L)

Total dead/immobile at 48 h

Total tested

Percentage immobility at 48 h

Control

0

10

0

0.01

1

10

10

0.1

3

10

30

1.0

2

10

20

10

10

10

100

100

10

10

100

Analytical results

Nominal concentration of Test substance

(mg/L)

Measured concentration of Test substance

(mg/L)

Mean measured concentration

(mg/L)

Mean measured concentration

(%)

0 h

48 h

(mg/L)

% of nominal

(mg/L)

% of nominal

Control

<LOQ

-

<LOQ

-

0

-

0.010

<LOQ

0

<LOQ

0

0

0

0.032

<LOQ

0

<LOQ

0

0

0

0.102

0.12

119

0.13

125

0.12

122

0.328

0.28a

86

0.44b

133

0.36

110

1.05

0.85

81

1.3

128

1.1

104

3.4

2.4

71

3.2

93

2.8

82

10.7

8.3

77

10

96

9.3

87

All measurements are quoted to 2 significant figures

a    Mean of triplicate analyses: 0.28, 0.29, 0.28 mg/L

b    Mean of triplicate analyses: 0.40, 0.48, 0.43 mg/L

The limit ofquantification (LOQ) in this study was 0.1 mg/L for all test solutions with the exception of the nominal 10.7 mg/L which was 0.2 mg/L. The instrument LOQ was 0.05 mg/L but during analysis, samples from the control and each test concentration were diluted x2, doubling the LOQ, with the exception of the nominal 10.7mg/L which was diluted x4. Values have been corrected for these dilutions. Where measured concentrations were less than the LOQ, zero values were used to calculate the mean.

Biological data

Daphnia magna response

Time

(h)

Nominal concentration of Test substance

(mg/L)

Mean measured concentration of Test substance

(mg/L)

Number immobilised per replicate

Total number tested

Total number immobilised

Percentage immobilised

A

B

C

D

24

Control

0

0

0

0

0

20

0

0

0.010

0

0

0

0

0

20

0

0

0.032

0

0

0

0

0

20

0

0

0.102

0.12

0

0

0

0

20

0

0

0.328

0.36

0

0

0

0

20

0

0

1.05

1.1

0

0

0

0

20

0

0

3.4

2.8

4

4

2

3

20

13

65

10.7

9.3

5

3

4

4

20

16

80

48

Control

0

0

0

0

0

20

0

0

0.010

0

0

0

0

0

20

0

0

0.032

0

0

0

0

0

20

0

0

0.102

0.12

0

0

0

0

20

0

0

0.328

0.36

0

0

0

0

20

0

0

1.05

1.1

0

0

2

0

20

2

10

3.4

2.8

4

4

2

3

20

13

65

10.7

9.3

5

4

4

4

20

17

85

 


The results obtained (based on mean measured concentrations of test substance) were:

Time

EC50(mg/L)

95% confidence limits

Calculation method

24 h

2.4

2.0-3.0

Linear interpolation (ICPIN)

48 h

2.3

1.7-3.1

Linear interpolation (ICPIN)

 

Based on immobility compared to the control (p <0.05) the 48 h No Observed Effect Concentration (NOEC) was determined to be 1.1 mg/L and the Lowest Observed Effect Concentration (LOEC) was 2.8 mg/L. There was no immobility observed in the dilution water control. No other symptoms of toxicity were observed.

Validity criteria

The OECD 202 Test Guideline details the following performance criteria for the test validity:

1)    In the control, no more than 10% of the daphnids should have been immobilised or show other signs of stress;

2)    The dissolved oxygen concentration at the end of the test should be 3.0 mg/L in control and test vessels.

3)    As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at 3 mg/L.

This test has satisfied all validity criteria.

Validity criteria fulfilled:
yes
Conclusions:
Under study conditions, the 48 h EC50 and NOEC value of the test substance were determined to be 2.3 mg/L and 1.1 mg/L (measured) respectively.
Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed each to nominal concentrations of 0, 0.010, 0.032, 0.102, 0.328, 1.05, 3.4 and 10.7 mg/L for 48 h under static conditions. The concentrations of the substance in the test vessels were measured using the high-performance liquid chromatography method. The mean measured concentrations of the substance were determined to be 0, 0 (0.010), 0 (0.032), 0.12, 0.36, 1.1, 2.8 and 9.3 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h No Observed Effect Concentration (NOEC) was determined to be 1.1 mg/L (measured) and the Lowest Observed Effect Concentration (LOEC) was 2.8 mg/L (measured). No other symptoms of toxicity were observed. The 48 h EC50 was calculated to be 2.3 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all validity criteria. Under the study conditions, the 48 h EC50 and NOEC value of the read across substance were determined to be 2.3 mg/L and 1.1 mg/L (measured) respectively (Scymaris, 2017). Based on the results of the read across study, a similar EC50 value can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'

Description of key information

Based on the results of the read across study, the 48 h EC50 value of the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE' is considered to be 2.3 mg/L (measured).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.3 mg/L

Additional information

A study was conducted to determine the acute toxicity of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Twenty test organisms were exposed each to nominal concentrations of 0, 0.010, 0.032, 0.102, 0.328, 1.05, 3.4 and 10.7 mg/L for 48 h under static conditions. The concentrations of the substance in the test vessels were measured using the high-performance liquid chromatography method. The mean measured concentrations of the substance were determined to be 0, 0 (0.010), 0 (0.032), 0.12, 0.36, 1.1, 2.8 and 9.3 mg/L. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on immobility compared to the control (p <0.05), the 48 h No Observed Effect Concentration (NOEC) was determined to be 1.1 mg/L (measured) and the Lowest Observed Effect Concentration (LOEC) was 2.8 mg/L (measured). No other symptoms of toxicity were observed. The 48 h EC50 was calculated to be 2.3 mg/L (measured). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at ≥3 mg/L, this test has satisfied all validity criteria. Under the study conditions, the 48 h EC50 and NOEC value of the read across substance were determined to be 2.3 mg/L and 1.1 mg/L (measured) respectively (Scymaris, 2017). Based on the results of the read across study, a similar EC50 value can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE'