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Ecotoxicological information

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:
The study was conducted between 18 May 2017 and 13 June 2017
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
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: RM1008356
Physical state/Appearance: White powder
Batch: 161221
Purity: 100%
Expiry Date: 13 February 2020
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 10, 14, and 19 and of the expired test preparations on Days 3, 7, 12, 17 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Samples at the No Observed Effect Loading Rate only were analyzed.
Vehicle:
no
Details on test solutions:
Validation of Mixing Period:
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
Initial solubility testing showed the test item to be insoluble using traditional methods of ultrasonication and high shear mixing. Therefore, given the complex and insoluble nature of the test item, a validation of mixing period trial was conducted.
It was evident from these results that there was no increase in the dissolved test item concentration obtained by extending the preparation period beyond 24 hours. Therefore the test item was prepared as a WAF using a 23-Hour stirring period followed by a 1-Hour settlement period prior to removal of the aqueous phase for testing.

Definitive Test:
Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of test concentrations of 26, 33, 41, 51, 64, 80 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week throughout the test.

Nominal amounts of test item (78, 99, 123, 153, 192, 240 and 300 mg) were each separately added to the surface of 3 liters of test water to give the 26, 33, 41, 51, 64, 80 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Previous observations made on WAF preparations with this test item indicated that a significant amount of dispersed test item was present in the water column and after discussion with the Sponsor it was considered justifiable, as a precaution, to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 26, 33, 41, 51, 64, 80 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions present.

The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 3, 5, 7, 10, 12, 14, 17, 19 and 21.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test water:
The reconstituted water (Elendt M7 medium) used for the definitive test was the same as that used to maintain the stock animals.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.
See water quality criteria results.
Test temperature:
The test vessels were maintained in a temperature controlled room at 18 to 22°C
See water quality criteria results.
pH:
pH was recorded before and after each test media renewal. See results section.
See water quality criteria results.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded before and after each test media renewal.
See water quality criteria results.
Nominal and measured concentrations:
Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 26, 33, 41, 51, 64, 80 and 100 mg/L.
Measured: See results.
Details on test conditions:
Exposure conditions:
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were renewed 3 times per week on Days 3, 5, 7, 10, 12, 14, 17 and 19. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.

Each daphnid received approximately 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding
was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Assessments:
Test Organism Observations:
On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.

The number of daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

At the end of the test, the length of each surviving parent animal was determined.

Water Quality Criteria:
Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in one replicate of the control using a Testo temperature logger. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.

Statistical analysis:
An estimate of the 21-Day EL50 value was given by inspection of the immobilization data.

The ELx (reproduction) values and associated confidence limits after 21 days were estimated by inspection of the data.

For the estimation of the "No Observed Effect Loading Rate " (NOEL) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using the Multiple Sequentially-rejective U-test After Bonferroni-Holm using ToxRat professional computer software package (ToxRat, 2015).

Results from the control and each test groups daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).









Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rates
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rates
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rates
Basis for effect:
other: immobilization and reproduction
Details on results:
Verifcation of Test Concentrations:
Chemical analysis of the freshly prepared test preparations on 0, 5, 10, 14 and19 showed measured test concentrations to range from less than the Limit of Qunatification (LOQ) of the analytical method, determined to be 0.044 mg/L, to 0.28 mg/L.
Chemical analysis of the aged test preparations on Days 3, 7, 12, 17 and 21 showed measured test concentrations to range from less than the LOQ to 0.12 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Test Organism Observations:
Lethal Effects on the Parental Generation (P1):
Mortality was observed at the test loading rate of 51 mg/L on Day 7 and Day 9. However, statistical analysis of the mortality data using the Fisher’s Exact test showed that the observed mortalities were not significantly different (P≥0.05) when compared to the control group.

No mortalities occurred in the 28, 33, 41, 64, 80 and 100 mg/L loading rate WAF groups throughout the test.

The ELx (immobilization) value based on nominal loading rates was estimated by inspection of the data:
Immobization EC10: >100 mg/L (nominal loading rate)
Immobization EC50: >100 mg/L (nominal loading rate)
No Observed Effect Loading Rate (NOEL): 100 mg/L (nominal loading rate)

Sub-lethal Effects on the Parental Generation (P1):
There was no significant effect on the size and color of the daphnids in all test loading rates throughout the test.
After 21 days the length of each surviving adult was determined. The results showed that there was a significant difference between the control and the 33, 41, 64 and 80 mg/L loading rates, however, no significant differences (P≥0.05) were found between the control and the 26, 51 and 100 mg/L loading rate groups. The differences seen were considered to be due to natural variation and as such were considered not to have an impact on the outcome of the test

Effects on Reproduction:
Analysis of the data obtained on Day 21 showed that there was no statistically significant difference (P≥0.05) between the control and the 26, 33, 41, 64, 80 and 100 mg/L loading rate test groups. A significant difference was found between the control and the 51 mg/L loading rate, however, given that no effect was observed in the higher concentrations, this was considered not to have had an impact on the outcome of the test.

The following ELx (reproduction) values based on nominal loading rates were estimated by inspection of the data:
Reproduction EC10: >100 mg/L (nominal loading rate)
Reproduction EC50: >100 mg/L (nominal loading rate)
No Observed Effect Loading Rate (NOEL): 100 mg/L (nominal loading rate)

Effects on the Filial Generation (F1):
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Young were first produced in the control test group on Day 9 of the test.

Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

No Observed Effect Loading Rate:
The "No Observed Effect Loading Rate" (NOEL) was 100 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Water Quality Criteria:
Temperature was maintained at approximately 20°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The water hardness was observed to be in the range 262 to 282 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.

Throughout the test the light intensity was observed to be in the range 414 to 647 lux

Vortex Depth Measurements:
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility:
Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of the mixing period the 26, 33, 41, 51, 64, 80 and 100 mg/L loading rates were observed to be clear colorless water columns with test item floating at the surface. After
23 hours stirring and a 1-Hour standing period the all loading rates were observed to remain as at the start of stirring. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present, however the loading rates were siphoned through a glass wool plug as a precaution. After siphoning and for the duration of the test, the control and all test loading rates were observed to be a clear, colorless solutions.

On Day 3 the 100 mg/L loading rate after 23 hours stirring and 1 hour settlement was observed to be a clear colorless water column with particles of test item dispersed throughout. After filtration through a glass wool plug the test solution was observed to be a clear colourless solution and microscopic observations showed no microdispersions visible.









Summary of Findings Following the Exposure of Daphnia magna for 21 days.

 

Nominal Loading Rate (WAF)

Parental (P1) Generation Mortalities (Initial Population = 10)

Total Number of Live Young

Number of Live Young from Parents Surviving to the End of the Test

Number of Live Young at the End of the Test Excluding Parents that were Accidental or Inadvertent Mortalities

Total

Accidental

Inadvertent

Total

Average per Parent

Total

Average per Parent

Control

1

0

1

1267

1128

128

1128

128

26

0

0

0

1138

1138

118

1138

118

33

0

0

0

1237

1237

124

1237

124

41

0

0

0

1221

1221

122

1221

122

51

2

0

2

939

931

116

931

116

64

1

0

1

1157

1056

117

1056

117

80

0

0

0

1231

1231

123

1231

123

100

0

0

0

1211

1211

121

1211

121

Summary of Observations.

 

 

Day

Nominal Loading Rate (mg/L)

Control

26

33

41

51

64

80

100

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

Adults Surviving

Live Young

1

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

2

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

3

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

4

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

5

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

6

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

0

7

10

0

10

0

10

15

10

0

9

8

10

0

10

0

10

5

8

10

0

10

49

10

49

10

31

9

27

10

27

10

72

10

44

9

10

36

10

0

10

13

10

50

8

24

10

34

10

45

10

21

10

10

69

10

15

10

36

10

14

8

23

10

42

10

2

10

31

11

10

0

10

79

10

68

10

46

8

35

10

41

10

120

10

60

12

10

145

10

51

10

18

10

63

8

26

10

103

10

0

10

35

13

10

0

10

0

10

131

10

71

8

90

10

42

10

0

10

56

14

10

33

10

169

10

153

10

107

8

105

10

54

10

301

10

158

15

10

290

10

84

10

0

10

109

8

40

10

125

10

0

10

53

16

10

32

10

41

10

214

10

100

8

155

10

62

10

0

10

78

17

10

59

10

222

10

76

10

187

8

58

10

132

10

339

10

216

18

10

262

10

61

10

90

10

87

8

54

10

137

10

1

10

67

19

10

3

10

165

10

212

10

112

8

167

10

142

10

129

10

124

20

9

136

10

115

10

44

10

151

8

83

10

81

10

222

10

187

21

9

202

10

87

10

118

10

93

8

44

9

135

10

0

10

76

TOTALS

 

1267

 

1138

 

1237

 

1221

 

939

 

1157

 

1231

 

1211

Total Cumulative Production of Live Young

Nominal Loading Rate (mg/L)

Day

1

2

3

4

5

6

7

8

9

10

Control

0

0

0

0

0

0

0

0

36

105

26

0

0

0

0

0

0

0

49

49

64

33

0

0

0

0

0

0

15

64

77

113

41

0

0

0

0

0

0

0

31

81

95

51

0

0

0

0

0

0

8

35

59

82

64

0

0

0

0

0

0

0

27

61

103

80

0

0

0

0

0

0

0

72

117

119

100

0

0

0

0

0

0

5

49

70

101

Nominal Loading Rate (mg/L)

Day

11

12

13

14

15

16

17

18

19

20

21

Control

105

250

250

283

573

605

664

926

929

1065

1267

26

143

194

194

363

447

488

710

771

936

1051

1138

33

181

199

330

483

483

697

773

863

1075

1119

1237

41

141

204

275

382

491

591

778

865

977

1128

1221

51

117

143

233

338

378

533

591

645

812

895

939

64

144

247

289

343

468

530

662

799

941

1022

1157

80

239

239

239

540

540

540

879

880

1009

1231

1231

100

161

196

252

410

463

541

757

824

948

1135

1211

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the loading rates employed during the test.

The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was calculated to be greater than 100 mg/L.

No significant impairment of reproduction was observed at the loading rates employed during the test.

The 21-Day EL50 (reproduction) based on nominal loading rates was greater than 100 mg/L.

The "No Observed Effect Loading Rate" (NOEL) based on nominal loading rates was 100 mg/L.
Executive summary:

Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 26, 33, 41, 51, 64, 80 and 100 mg/L for a period of 21 days.  The test solutions were renewed 3 times per week throughout the test.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Results

Chemical analysis of the 100 mg/L loading rate fresh test preparations on Days 0, 5, 10, 14 and 19 showed measured test concentrations to range from less than the Limit of Quantification (LOQ), determined to be 0.044 mg/L, to 0.28 mg/L. Chemical analysis of the aged test preparations on Days 3, 7, 12, 17 and 21 showed measured test concentrations to range from less than the LOQ to 0.12 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental Daphnia generation (P1) was calculated to be greater than 100 mg/L loading rate WAF.

The 21-Day EL50 (reproduction) value, based on nominal loading rates, was calculated to be greater than 100 mg/L loading rate WAF.

The "No Observed Effect Loading Rate" was considered to be 100 mg/L on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences between the control and the 100 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Description of key information

Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 26, 33, 41, 51, 64, 80 and 100 mg/L for a period of 21 days.  The test solutions were renewed 3 times per week throughout the test.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Results

Chemical analysis of the 100 mg/L loading rate fresh test preparations on Days 0, 5, 10, 14 and 19 showed measured test concentrations to range from less than the Limit of Quantification (LOQ), determined to be 0.044 mg/L, to 0.28 mg/L. Chemical analysis of the aged test preparations on Days 3, 7, 12, 17 and 21 showed measured test concentrations to range from less than the LOQ to 0.12 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

The 21-Day EL50(immobilization) value, based on nominal loading rates, for the parental Daphnia generation (P1) was calculated to be greater than 100 mg/L loading rate WAF.

The 21-Day EL50(reproduction) value, based on nominal loading rates, was calculated to be greater than 100 mg/L loading rate WAF.

The "No Observed Effect Loading Rate" was considered to be 100 mg/L on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences between the control and the 100 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Key value for chemical safety assessment

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

Additional information