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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:
August 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
see details below
Principles of method if other than guideline:
Due to a data logger failure, the test temperatures were not recorded throughout all the test period of the validated definitive test, only the three last days of the test period are reported. Nevertheless, the temperature is supposed to have been maintained since the experiment is done in climatic chamber under metrological control. In addition, the validity criteria have been met for the control group.
This deviation was not considered to have affected the outcome or the achievement of the study objectives.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control on 4 occasions throughout the test, on fresh and old solutions as follows:
- at days 0 (fresh solutions) and 1 (old solutions)
- at days 7 (fresh solutions) and 8 (old solutions)
- at days 14 (fresh solutions) and 15 (old solutions)
- at days 19 (fresh solutions) and 20 (old solutions)
Vehicle:
no
Details on test solutions:
For both range-finding test and definitive tests, as the test item was known to be difficult to dissolve in water, saturated stock solutions were prepared using slow-stirring conditions as described in the OECD 123 guideline. Due to daily renewal, a new test item stock solution was prepared daily. The method used special temperature controlled glass-jacketed test vessels (around 1000 mL of capacity), with a sampling tap at the lower end and a magnetic stir bar at its bottom.

A known volume of test medium (around 1000 mL) was first poured into the flask then a known quantity of test item (106.8 µL) was gently added at the surface of medium in order to reach a loading rate of around 100 mg/L. The solution was stirred at room temperature with a magnetic stir bar at a speed avoiding formation of an emulsion. A speed of approximately 250-300 rpm was set up in order to create a vortex depth of around 0.5 cm: the aqueous phase was kept under stirring during approximately 72 hours then drawn off as a clear solution after a rest phase of 30 minutes to 1h without agitation. Test solutions were then prepared from dilutions of this saturated solution.
The pH of the test solutions were not adjusted.

PRELIMINARY RANGE-FINDING TEST
After consultation with the Sponsor’s Representative, the preliminary range-finding test was conducted at the nominal test item concentrations of 0.32, 1.03 and 3.30 mg/L, together with a control group. Test concentrations were arranged in a geometric series with a separation factor of 3.2. Five replicate vessels were prepared at each concentration including the control group.

At the start of the experiment one D. magna neonate, less than one day old, was added to each vessel. Daphnia were fed as described above. The total test period was 13 days. All test solutions, including control were renewed every day, including weekends.

Samples for analysis were taken from all test concentrations and the control on 2 occasions throughout the test, on fresh and old solutions as follows:
- at days 0 (fresh solutions) and 1 (old solutions)
- at days 4 (fresh solutions) and 5 (old solutions)

DEFINITIVE REPRODUCTION TEST
After consultation with the Sponsor’s Representative, the reproduction test was conducted at the nominal test item concentrations of 0.02, 0.06, 0.15, 0.38 and 1.00 mg/L, together with a control group. Test concentrations were arranged in a geometric series with a separation factor of 2.6. Ten replicate vessels were prepared at each concentration including the control group.

At the start of the experiment one D. magna neonate, less than one day old, was added to each vessel. Daphnia were fed as described above. The total test period was 21 days. All test solutions, including control, were renewed every day, including weekends.



Test organisms (species):
Daphnia magna
Details on test organisms:
The test organisms used for this study was Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. D. magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny.

Test animals were fed a diet of around 0.1 - 0.2 mg of carbon per daphnid per day, in the form of a suspension of the algal strains Chlorella vulgaris (25%) and Pseudokirchneriella subcapitata (75%), except during the initial three to five days of culture when a slightly lower ration was given. The concentrated algal cell suspension was prepared by removing and centrifuging aliquots of algal culture and resuspending the algal pellets in small volumes of dilution medium.

All testing was performed using a synthetic medium prepared with Volvic® water and complemented as follows:
• 0.1 ml/L of B12 vitamin solution (1 µg/L)
• 0.1 ml/L of Na2SeO3.5H2O solution (6.7 µg/L)
• 1 ml/L of calcium / magnesium solution: Ca(NO3)2.4H2O : 208 mg/L and MgCl2.6H2O : 28 mg/L.

Chemical reagents used for the preparation of this dilution water were of “analytical grade”.

Test vessels containing D. magna were incubated in climatic chamber at a temperature ranging from 18°C to 22°C but remaining constant within ± 2°C during the test. A light cycle of 16 h light and 8 h dark was in operation throughout the test, provided by LED light (light intensity around 800 lux).

Test vessels were made of clear glass tubes (120 mL capacity) and stoppered with cellulose bungs to minimise evaporation and avoid the entry of dust into the solutions. At least 50 mL of exposure solution was provided for each organism.

The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item. No auxiliary substance was used.

Every day, all parent animals were observed for immobilisation by gentle agitation of the test vessel. Any immobile animal within 15 seconds following agitation was considered as dead and discarded. The surviving parental Daphnia were transferred into fresh control or test media in a second set of vessels by means of a wide bore glass pipette. From the day of the first brood, all neonates (dead and alive), unhatched eggs or carapaces present in the expired media were counted daily and discarded. Any other abnormal observation was also recorded and reported.

pH and dissolved oxygen were measured in the fresh and old solutions in one of the vessels of each test group with surviving daphnids.

The total hardness used to prepare test solutions was determined using a Hardness test kit (MColortest®, Merck).

The temperature was recorded in a flask maintained within the same conditions.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
The total hardness (as CaCo3) of the test medium used to prepare test solutions was determined on fresh solutions using a Hardness test kit on days 0, 7 and 14: 175-180 mg/L CaCO3
Test temperature:
Temperature was measured as follows: min. 20.1, max. 20.3°C and mean: 20.2°C
pH:
Fresh solutions: 7.4-7.9
Old solutions: 7.9-8.2
Dissolved oxygen:
Fresh solutions: 8.6-9.5 mg/L
Old solutions: 8.6-9.4 mg/L
Salinity:
NA
Nominal and measured concentrations:
Nominal concentrations: 0, 0.02, 0.06, 0.15, 0.38 and 1.00 mg/L
see details of measured concentrations below.
Details on test conditions:
Light intensity measured was between 480 and 997 lux.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
483.4 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
168.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
PRELIMINARY RANGE-FINDING TEST
All test solutions appeared clear and colourless. No precipitation of test item nor undissolved particles were observed. Chemical analysis of the test item showed the instability of the substance over a 24-hour period.
There were no immobile Daphnia magna in the control group and in all treatment groups.
Test solution quality parameters (pH and dissolved oxygen concentration) were measured in one replicate vessel at each concentration. Parameters remained within the following ranges: pH: 7.4 - 8.7 and dissolved oxygen concentration: 8.9 - 10.9 mg/L.
Temperature was measured as follows: min. 20.1, max. 20.4°C and mean: 20.3°C, and light intensity measured at 650 to 850 lux

DEFINITIVE REPRODUCTION TEST
All test solutions appeared clear and colourless. No precipitation of test item nor undissolved particles were observed. Chemical analysis of the test item showed the instability of the substance over a 24-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.
There were no immobile Daphnia magna in the control group and in all treatment groups.
Test solution quality parameters (pH and dissolved oxygen concentration) were measured in one replicate vessel at each concentration. Parameters remained within the following ranges: pH: 7.4 - 8.2 and dissolved oxygen concentration: 8.5 - 9.5 mg/L.
The total hardness (as CaCo3) of the test medium used to prepare test solutions was determined on fresh solutions using a Hardness test kit on days 0, 7 and 14.
Temperature was measured as follows: min. 20.1, max. 20.3°C and mean: 20.2°C (see paragraph 1.6), and light intensity measured between 480 and 997 lux.

Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated monthly by performing an acute Daphnia magna toxicity test on potassium dichromate. The nearest value of 24h-EC50 obtained on January 2018 was 0.74 mg/L.
For information, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.
Reported statistics and error estimates:
Statistical analysis was performed with the software ToxRat V 3.2.1. See statistical report Appendix 4.

Summary of the biological data obtained during the preliminary range-finding test

 

Control

0.32

1.03

3.30

Total of living offspring (per parent alive)*

205

140

81

0

Mean of living offspring produced per parent

41.0

28.0

16.2

0.0

Standard Deviation

22.2

16.4

10.6

0.0

Total of dead offsprings

7

21

18

0

Total of eggs

3

2

2

0

Total of dead parents

0

0

0

0

* produced at the end of the test by all the parent daphnia integrated at the start of the test excluding parental accidental and/or inadvertent mortality

Preliminary range-finding test: test item measured concentrations

Nominal conc.

(mg/L)

Time

(days)

fresh

(µg/L)

old

(µg/L)

 Deviationof old Test Conc. from fresh Test Conc. (%)

Control

0

<DL

-

NA

1

-

<DL

4

<DL

-

NA

5

-

<DL

0.32

0

345

-

-68.3

1

-

110

4

252

-

-71.6

5

-

71.6

1.0.

0

1099

-

-66.3

1

-

371

4

817

-

-59.1

5

-

334

3.30

0

3664

-

-67.2

1

-

1203

4

3002

-

-59.8

5

-

1208

NA: not applicable

< DL: concentration lower than the Detection Limit (5.6 µg/L)

< QL: concentration lower than the Quantification Limit (18.4 µg/L)

Summary of the biological data obtained during the definitive reproduction test

 

Control

0.02

0.06

0.15

0.38

1.00

Total of living offspring (per parent alive)*

1154

1297

1129

1261

1259

795

Mean of living offspring produced per parent

115.4

129.7

112.9

126.1

125.9

79.5

Standard Deviation

28.0

20.7

19.4

12.2

12.1

14.2

Total of dead offspring

7

7

8

2

8

4

Total of eggs

10

14

12

15

15

8

Total of dead parents

0

0

0

0

0

0

* produced at the end of the test by all the parent daphnia integrated at the start of the test excluding parental accidental and/or inadvertent mortality

Reproduction test: test item measured concentrations

# occasion

Sample

Nominal Conc.

(mg/L)

Test item conc. (µg/L)

RSD

(%)

Measure 1

Measure 2

Measure 3

Mean

SD

1

Fresh

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<QL

<QL

<QL

<QL

NA

NA

0.06

31.1

31.5

33.8

32.1

1.5

4.5

0.15

110.5

105.9

111.4

109.3

3.0

2.7

0.38

330.4

338.0

333.1

333.8

3.9

1.2

1.00

944.5

942.9

951.9

946.4

4.8

0.5

Old

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<DL

<DL

<DL

<DL

NA

NA

0.06

<DL

<QL

<QL

NA

NA

NA

0.15

29.9

30.3

28.8

29.7

0.8

2.6

0.38

103.3

100.6

103.8

102.6

1.7

1.7

1.00

307.2

301.9

303.2

304.1

2.8

0.9

2

Fresh

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<QL

<QL

<QL

<QL

NA

NA

0.06

29.3

27.5

26.3

27.7

1.5

5.3

0.15

94.0

88.9

96.8

93.2

4.0

4.3

0.38

285.2

306.2

299.9

297.1

10.8

3.6

1.00

846.8

856.2

898.4

867.1

27.5

3.2

Old

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<DL

<DL

<DL

<DL

NA

NA

0.06

<QL

<QL

<QL

<QL

NA

NA

0.15

28.8

29.8

27.6

28.7

1.1

3.8

0.38

75.9

76.4

80.6

77.6

2.6

3.4

1.00

213.2

213.3

219.0

215.2

3.3

1.5

3

Fresh

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<QL

<QL

<QL

<QL

NA

NA

0.06

47.5

47.1

44.7

46.4

1.5

3.3

0.15

129.3

129.5

138.2

132.3

5.1

3.8

0.38

374.0

378.6

379.4

377.3

2.9

0.8

1.00

1079.3

1084.3

1087.5

1083.7

4.1

0.4

Old

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<DL

<DL

<DL

<DL

NA

NA

0.06

<DL

<QL

<QL

NA

NA

NA

0.15

22.2

<QL

<QL

NA

NA

NA

0.38

66.7

71.7

72.0

70.1

3.0

4.2

1.00

191.7

188.5

188.2

189.5

1.9

1.0

4

Fresh

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<QL

<QL

<QL

<QL

NA

NA

0.06

44.0

39.4

43.9

42.5

2.6

6.2

0.15

128.4

129.7

132.2

130.1

1.9

1.5

0.38

336.5

333.9

334.8

335.1

1.3

0.4

1.00

975.7

981.0

981.7

979.5

3.3

0.3

Old

Control

<DL

<DL

<DL

<DL

NA

NA

0.02

<DL

<DL

<DL

<DL

NA

NA

0.06

<QL

<QL

<QL

<QL

NA

NA

0.15

27.4

34.0

28.0

29.8

3.6

12.2

0.38

96.1

95.3

91.8

94.4

2.3

2.4

1.00

277.3

274.9

276.2

276.1

1.2

0.4

NA: not applicable

< DL: concentration lower than the Detection Limit (5.8 µg/L)

< QL: concentration lower than the Quantification Limit (19.4 µg/L)

Reproduction test: concentrations used for statistical analysis

Nominal Conc.

(mg/L)

Test item measured concentrations(µg/L)

Used concentrations* (µg/L)

Occasion #1

Occasion #2

Occasion #3

Occasion #4

fresh

old

Deviation

fresh

old

Deviation

fresh

old

Deviation

fresh

old

Deviation

Control

<DL

<DL

NA

<DL

<DL

NA

<DL

<DL

NA

<DL

<DL

NA

NA

0.02

9.7

5.8

-40.2

9.7

5.8

-40.2

9.7

5.8

-40.2

9.7

5.8

-40.2

7.5

0.06

32.1

8.4

-73.9

27.7

9.7

-65.0

46.4

8.4

-81.9

42.4

9.7

-77.1

18.1

0.15

109.3

29.7

-72.8

93.2

28.7

-69.2

132.3

13.9

-89.5

130.1

29.8

-77.1

52.9

0.38

333.8

102.6

-69.3

297.1

77.6

-73.9

377.3

70.1

-81.4

335.1

94.4

-71.8

168.9

1.00

946.4

304.1

-67.9

867.1

215.2

-75.2

1083.7

189.5

-82.5

979.5

276.1

-71.8

483.4

NA: not applicable

< DL: concentration lower than the Detection Limit (5.8 µg/L)

< QL: concentration lower than the Quantification Limit (19.4 µg/L)

When test item measured concentration is < DL. DL value was used

When test item measured concentration is < QL. half QL value was used

*Concentrations extrapolated as geometric mean of all measured concentrations

Validity criteria fulfilled:
yes
Remarks:
see details in the Conclusions section
Conclusions:
The lowest NOEC was observed for the mean number of neonates produced per surviving parent. This value of NOEC for the test item was 168.9 µg/L, expressed as geometric means of measured concentrations.

The definitive test met the validity criteria of the test guideline detailed as follows (for the control group):
- the mortality of the parent animals (female Daphnia) did not exceed 20% at the end of the test (0%);
- the mean number of living neonates produced per parent animal surviving at the end of the test is > 60 (115.4)
In addition the CV (24.3%) of the mean number of living neonates produced per control parent animal surviving at the end of the test was lower than the threshold recommended by the OECD 211 guideline (25%).
Executive summary:

This study was designed to determine the effects of tert-butyl cumyl peroxide on Daphnia magna reproduction and survival in a 21 -day test according to the OECD 211 Guideline (2012) and according to GLP requirements.  Daphnia magna were exposed to solutions of the test item at nominal concentrations of 0.02, 0.06, 0.15, 0.38 and 1.00 mg/L, together with a control group. The test solutions (including control) were renewed daily throughout the test, including weekends. The test solutions were prepared by gently adding the test item at the surface of the test medium to obtain a loading rate of around 100 mg/L. The solution was stirred during approximately 72 hours then drawnoff as a clear solution after a rest phase of 30 min-1h without agitation.This saturated solution was then further diluted to obtain each test solution. Ten replicate test vessels were prepared for the control and for each test concentration. A single juvenile Daphnia magna (<24 hours old selected by filtration) was added to each test vessel. On renewal occasion, parental animals were transferred into freshly prepared test medium. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on any day during the 21-day study duration was recorded.The time of the first production of young, the number of young born, immobility and other signs of toxicity observed were compared to a control group.

Chemical analysis was made on fresh and old solutions throughout the test. A decline in measured test concentration of the aged test solutions was observed. Based on these results, the exposure concentrations were based on the geometric means of measured concentrations. NOEC and LOEC after 21 days of exposure were calculated to be as follows:

Endpoint

NOEC

LOEC

Mean number of neonates produced per survivor

168.9 µg/L

483.4 µg/L

Endpoint:Age at first brood

 483.4 µg/L

> 483.4 µg/L

There were no immobile parent Daphnia magna in the control group and in all treatment groups. In this study, the lowest NOEC (168.9 µg/L) was observed for the mean number of neonates produced per surviving parent.

The definitive test met all the validity criteria of the test guideline and thus this experiment has been considered as valid.

Description of key information

The chronic toxicity to aquatic invertebrates was assessed according to the OECD Guideline 211 and GLP requirements. The lowest NOEC was observed for the mean number of neonates produced per surviving parent. This value of NOEC for the test item was 168.9 µg/L, expressed as geometric means of measured concentrations.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
168.9 µg/L

Additional information

This study was designed to determine the effects of tert-butyl cumyl peroxide on Daphnia magna reproduction and survival in a 21 -day test according to the OECD 211 Guideline (2012) and according to GLP requirements (Chastenet, 2018). Daphnia magna were exposed to solutions of the test item at nominal concentrations of 0.02, 0.06, 0.15, 0.38 and 1.00 mg/L, together with a control group. The test solutions (including control) were renewed daily throughout the test, including weekends. The test solutions were prepared by gently adding the test item at the surface of the test medium to obtain a loading rate of around 100 mg/L. The solution was stirred during approximately 72 hours then drawnoff as a clear solution after a rest phase of 30 min-1h without agitation.This saturated solution was then further diluted to obtain each test solution. Ten replicate test vessels were prepared for the control and for each test concentration. A single juvenile Daphnia magna (<24 hours old selected by filtration) was added to each test vessel. On renewal occasion, parental animals were transferred into freshly prepared test medium. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on any day during the 21-day study duration was recorded.The time of the first production of young, the number of young born, immobility and other signs of toxicity observed were compared to a control group.

Chemical analysis was made on fresh and old solutions throughout the test. A decline in measured test concentration of the aged test solutions was observed. Based on these results, the exposure concentrations were based on the geometric means of measured concentrations. NOEC and LOEC after 21 days of exposure were calculated to be as follows:

Endpoint

NOEC

LOEC

Mean number of neonates produced per survivor

168.9 µg/L

483.4 µg/L

Endpoint:Age at first brood

 483.4 µg/L

> 483.4 µg/L

There were no immobile parent Daphnia magna in the control group and in all treatment groups. In this study, the lowest NOEC (168.9 µg/L) was observed for the mean number of neonates produced per surviving parent.

The definitive test met all the validity criteria of the test guideline and thus this experiment has been considered as valid.