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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July 2013 to 5 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
Due to the low solubility of the test substance, a water soluble fraction (WSF) of the stock concentrate was prepared prior to test solution preparation, by filtering the stock concentrate through a 0.2 μm cellulose filter (micropore). The water soluble fraction of test substance was subsequently diluted with dilution water media to achieve the series of test substance solutions tested.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Chemical analyses were performed at day 0 on the freshly prepared solutions (ON) and the corresponding old solutions prior to renewal (OFF) on day 3. Thereafter, the solutions were analysed once per week (ON and OFF) for the remainder of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A 2L stock concentrate of the test substance was prepared prior to the day of use in the dilution media. The concentration of the stock concentrate was an initial loading of 100 mg/L and was prepared in a glass conical flask. The stock concentrate was stirred over night using a PTFE magnetic follower at a temperature of 20 ± 2 ºC. Due to the low solubility of the test substance, a WSF of the stock concentrate was prepared prior to test solution preparation, by filtering the stock concentrate through a 0.2 μm cellulose filter (micropore).
The water soluble fraction of test substance was subsequently diluted with dilution water media to concentrations of an initial loading of 10, 18, 32, 56 and the water soluble fraction was tested as the top concentration of an initial loading of 100 mg/L. The WSFs were prepared the day before use and were stirred over night prior to preparation of the test solutions.
A dilution water control was used plus a dilution water control containing no Na2SiO3.9H2O for comparison with the minimal media. The control solutions were treated identically to the test substance. After filtration, the test solutions were prepared by direct addition of an appropriate volume of each WSF to fresh dilution water media. Fresh stock concentrates and test solutions were prepared 3 times per week.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Straus
- Source: continuous culture at NIVA
- Age: < 24 hours old
- Feeding: daphnids were fed on an algal diet consisting of Pseudokirchneriella subcapitata and supplemented with yeast
- Amount: the aounts fed were calculated to equate to between 0.1 and 0.2 mg carbon/daphnid/day (based on measurements of TOC)
- Frequency: daily with the exception of two occassions when the daphnids were fed twice the daily ration on the previous day to ensure that a sufficient amount of feed was received.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Not recorded.
Test temperature:
19.5 - 20.9 °C
pH:
7.64 - 8.98
Dissolved oxygen:
5.10 - 12.19 mg/L
Nominal and measured concentrations:
0, 10, 18, 32, 56, 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL plastic beakers
- Type (delete if not applicable): closed (test vessels were covered with a plastic lid)
- Fill volume: 50 mL
- Aeration: no
- Renewal rate of test solution: Fresh stock concentrates and test solutions were prepared 3 times per week.
- No. of organisms per vessel: 1 (P0) daphnid per vessel
- No. of vessels per concentration (replicates): 10
- No. of vessels per dilution water control (replicates): 10
- No. of vessels per dilution water control containing no Na2SiO3.9H2O (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The standard test media used for Daphnia reproduction tests are Elendt’s M4 or M7 medium, however, according to OECD 211, these media are not suitable for testing with chemicals containing metals due to its chelating properties. Therefore, the US EPA media was used but was supplemented with Na2SiO3.9H2O based on the previous findings of Lillicrap et al. (2014) where increased reproduction was observed in daphnids exposed to EPA media containing Silica fume. An EPA media only control (containing no Na2SiO3.9H2O) was also used for the study.
- Composition of EPA, 1993 media: 60 mg/L CaSO4.2H2O, 60 mg/L MgSO4.7H2O, 4 mg/L KCl, 96 mg/L NaHCO3, 10 mg/L Na2SiO3.9H2O.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light / 8 hours dark

EFFECT PARAMETERS MEASURED
Observations of mortalities were performed on a daily basis and an animal was recorded as dead if it was immobile, i.e. no observed movement of appendages or postabdomen within 15 seconds after gentle agitation of the test container. Other symptoms of toxicity that were observed were recorded. Observations were also made daily for the presence of offspring (termed the F1 generation) in each vessel.
The number of live offspring (F1 generation daphnids) and the presence of dead offspring or aborted eggs/embryos, in each test vessel were recorded on each day of solution renewal. At the end of the test, the surviving P0 daphnids were terminated and then measured for length using a microscope with an eyepiece graticule previously calibrated with a stage micrometer. The measurement of length was based on the distance between the apex of the helmet of the daphnids to the base of spine.

PHYSICOCEMICAL ANALYSIS
The pH and dissolved oxygen (DO) concentrations of the newly prepared control(s) and the highest test solution were measured using the excess solutions after filling the test vessels. The pH and dissolved oxygen concentration of a single replicate of the old control(s) and highest test solution were measured after transfer of the P0 daphnids.
Temperature was measured using a thermometer in a spare vessel and the light intensity was measured once during the test.

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality, reproduction and growth
Details on results:
SURVIVAL
There were no effects on the survival of the daphnids at any concentration of the test substances during the study. Furthermore, the validity criteria for survival of the control organisms (<20% mortality) was fulfilled during the study.

REPRODUCTION
There was no statistically significant reduction in daphnia offspring production at any concentration tested when compared to the control.

GROWTH
There were no significant reductions in the length of the daphnids at any concentration of test substance investigated.
Reported statistics and error estimates:
The analyses of the reproduction data generated in the study were based on the total number of living offspring produced per parent animal alive at the end of the test; therefore F1 daphnids produced by adults that died during the test were excluded from the calculations. The length and reproduction data were analysed using a multiple comparison procedure to identify the test concentrations which were significantly different from the control (P=0.05).

Table 1: Summary of the concentration of Mn (mg/L) measured during the study

Test concentration

(mg/L)

Mean measured concentration (ON) (mg/L)

Standard deviation

Mean measured concentration (OFF) (mg/L)

Standard deviation

0 (HRW)

0.0003

0

0.001

0.0006

0 (DWC)

0.0004

0.0001

0.0019

0.001

10

0.0044

0.0014

0.0065

0.003

18

0.0086

0.0017

0.0124

0.0053

32

0.0133

0.004

0.0193

0.011

56

0.0239

0.0061

0.0328

0.018

100

0.0408

0.0118

0.0568

0.0354

HRW - DWC without Si

Table 2: Summary of the biological data generated during the study

Test concentration (mg/L)

Mean no of offspring produced

Standard deviation

Mean length of daphnids (mm)

Standard deviation

0 (HRW)

95

19.3

4.26

0.14

0 (DWC)

102

14.7

4.26

0.19

10

99

17.9

4.28

0.12

18

91

26.2

4.19

0.19

32

109

25.6

4.15

0.21

56

89

25.1

4.03

0.21

100

108

18.9

4.33

0.15

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the 21-day No Observed Effect Concentration (NOEC) was determined to be 100 mg/L (based on nominal values).
Executive summary:

The chronic toxicity of the test substance to freshwater invertebrates was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 211.

During the study Dahnia magna, < 24 hours old, were exposed to a range of concentrations of test substance prepared from a water soluble fraction based on an initial loading of 100 mg/L. The test was performed at a temperature of 20 ± 2°C under semi-static test conditions with renewal of the test medium. The test duration was 21 days. Daphnia exposed concurrently in daphnia media only, served as the control. Mortality, immobility, time to first production of young and number of young produced and length after 21 days were recorded during the study.

The data generated during the study indicated that Mn3O4 did not cause a reduction in the number of offspring produced or affected the growth (length) of the organisms when compared to the control organisms after 21 days exposure. Therefore the NOEC for Daphnia magna reproduction and growth (length) for Mn3O4 is at an initial loading concentration of 100 mg/L. In addition, there were no effects of Mn3O4 on the survival of the daphnids during the study up to and including an initial loading of 100 mg/L. In conclusion, the NOEC of Mn3O4 on Daphnia magna should be expressed as an initial loading of 100 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2010-03-17 to 2010-04-12
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A number of deficiencies have been noted, as follows: The study duration was 8 days rather than 21 days; the test medium (Elendt M4) was not appropriate for testing metals (as stipulated in the OECD 211 guideline); and, furthermore, the levels of manganese measured in solution were below the limit of detection. Therefore, an environmental reference value cannot be obtained using results from this study. The findings from this study are therefore not useful and are disregarded in favour of findings of a 21-day study (Lilliput, 2013).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(see Principles of method if other than guideline)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
yes
Remarks:
(see Principles of method if other than guideline)
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess of test material in reconstituted water for a period of 48 hours prior to removing any undissolved test material present by filtration to give a saturated solution of the test material. Furthermore, the test organisms were Ceriodaphnia dubai rather than Daphnia magna. And the test duration was 8 days rather than 21 days.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of the test material in the test preparations was verified by chemical analysis on Day 0 (fresh media), 3, 5, 7 (old and fresh media) and 8 (old media).
- Sampling method: The test samples were analysed following addition of nitric acid (2.5 mL per 50 mL of sample).
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (250 mg) was dispersed in 2.5 litres of reconstituted water (Elendt M4) with the aid of magnetic stirring at approximately 100 rpm at approximately 25°C. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Gelman AcroCap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give the 100% v/v saturated solution. Aliquots (50 and 160 ml) of the 100% v/v saturated solution were each separately diluted in a final volume of 500 mL of reconstituted water (Elendt M4) to give the remainder of the test series of 10 and 32% v/v saturated solution.

Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Justification for species other than prescribed by test guideline: Ceriodapnia dubia is a freshwater invertebrate representative of a wide variety of natural habitats and can therefore be considered as an important non-target organism in freshwater ecosystems.
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD): 24 hours old
- Feeding during test: Each culture was fed daily
- Food type: Fed a suspension of algae (Pseudokirchneriella subcapitata) and YAT (yeast, alfalfa, trout chow) combination. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Amount: Each daphnid received 1µL to 5µL of a unicellular algal culture and 100 µl of a YAT combination. The study plan stated that each daphnid would be fed 1 µL of algal suspension daily. During the test each daphnid was fed 1 to 5 µL of algal suspension. This was due to a revised method of feeding whereby the amount of algal suspension fed was based on the cell density of the suspension. This deviation was considered not to have affected the outcome of the test.
- Frequency: Daily

ACCLIMATION
- Acclimation conditions: same as test
- Type and amount of food: same as test
- Feeding frequency: Fed daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
8 d
Hardness:
210 and 250 mg/L as CaCO3 in the control and the 100% v/v saturated solution test group at the start of the test
Test temperature:
25°C
pH:
8.0-8.3
Dissolved oxygen:
7.1 - 9.3 mg O2/L
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%.
Salinity:
Not reported
Nominal and measured concentrations:
Control, 10, 30 & 100 % saturated solution - Nominal concentration
Control, 0.52, 1.7 & 5.1 mg/L as test item - Mean measured concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): closed. Covered with a plastic lid to reduce evaporation
- Fill volume: 50 mL
- Aeration: No
- Renewal rate of test solution (frequency): The test preparations were renewed 3 times on Day 2, 3, and 7
- No. of organisms per vessel: 1
- 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 (Elendt M4) used for the definitive test was the same as that used to maintain the stock animals.
- Alkalinity: 48 and 50 mg CaCO3/L in the control and the 100% v/v saturated solution test group at the start of the test.
- Conductivity: 902 and 906 µs/cmin the control and the 100% v/v saturated solution test group at the start of the test.
- Intervals of water quality measurement: Temperature of the test preparations was recorded twice daily in two different locations and light intensity was recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on an additional sample (approximately 100 mL) of each test concentration run alongside the test due to the small volumes used in the test. The water hardness, conductivity and alkalinity of the control and the highest test concentration were determined at the start of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 8 days
- Light intensity: 551 to 581 lux

EFFECT PARAMETERS MEASURED
On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Ceriodaphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Ceriodaphnia as compared with the controls. The number of Ceriodaphnia 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 Ceriodaphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Based on the results of an acute toxicity test to Daphnia magna (Harlan Laboratories Ltd Project Number 2702/0161).
Reference substance (positive control):
no
Duration:
8 d
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 100% v/v saturated solution as this test group produced significantly fewer live young per adult (P<0.05) than the control.
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
32 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Mortality(immobilisation)
Remarks on result:
other: There were no significant mortalities (immobilisation) 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 8 days.
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: Parental Ceriodaphnia generation (P1).
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
78 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: Mortality (immobilisation) occurred predominantly at the highest test concentration of 100% v/v saturated solution, resulting in 40% mortality by Day 8, indicating a prolonged toxic effect attributable to exposure of Ceriodaphnia dubia to the test material.
Mortality was also observed at the test concentration of 32% v/v saturated solution. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortality in the 32% v/v saturated solution test group was not significantly different (P > 0.05) when compared to the control group. See table 2.

- No. of offspring produced per day per female: There was a significant effect on size and colour of the daphnids in that 33% of the surviving daphnids on Day 8 at the test concentration of 100% v/v saturated solution were markedly smaller and paler in colour than the control animals.
The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.
After 8 days there were no statistically significant differences between the control, 10 and 32% v/v saturated solution test groups in terms of the number of live young produced per adult. The 100% v/v saturated solution test group showed a statistically significant difference from the control and the remaining test groups after 8 days in terms of producing fewer numbers of live young per adult (see Appendix 4).
The EC50 (reproduction) value calculated by the maximum-likelihood probit method (Finney 1971) on Day 8, based on nominal test concentrations was 78% v/v saturated solution.

Information on the effects of the test material 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 group on Day 3 of the test. Numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation.


Reported statistics and error estimates:
See Any other information on material and methods incl.tables for details.
Table 2: The following EC50 (immobilisation) values based on nominal test concentrations as test material were estimated throughout the test

Time

EC50
(% v/v Saturated Solution)

24 hours

>100

48 hours

>100

96 hours

>100

8 days

>100*


*   It was not possible to calculate an EC50 value at this time point as less than 50% immobilisation occurred.

Table 3: Summary of findings following the exposure of Ceriodaphnia dubia for 8 days

Nominal Concentration (% v/v saturated solution)

% Survival of P1

No. of live young*

No. of dead young

No. of unhatched young           

Total

Per female*(cumulative)

Total

Per female (cumulative)

Total

Per female (cumulative)

Control

90

245

27

0

0

0

0

10

100**

214

24

0

0

0

0

32

90

193

21

0

0

0

0

100

60

62

10

0

0

0

0

* The number of live young per live adult

** A single live daphnid failed to produce any young throughout the duration of the test. This was considered to be possibly due to an inferior daphnid given the results from the remaining daphnids in the group. The results from this daphnid were therefore not included in the statistical analysis of the young produced

 

 

Table 4: Summary of observations of the control group

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

5

0

0

0

3

10

9

13

0

0

4

10

9

15

0

0

5

10

9

48

0

0

6

9

9

40

0

0

7

10

9

74

0

0

8

10

9

55

0

0

 

Total

245

0

0

 

 

Table 5: Summary of observations of the 10% v/v saturated solution test group

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

3

0

0

0

3

10

8

11

0

0

4

9

9

13

0

0

5

9

9

45

0

0

6

9

9

55

0

0

7

9

9

59

0

0

8

9

9

31

0

0

 

Total

214

0

0

 

Table 6: Summary of observations of the 32% v/v saturated solution test group

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

10

4

0

0

0

3

10

8

12

0

0

4

10

10

8

0

0

5

10

10

55

0

0

6

10

10

46

0

0

7

10

9

35

0

0

8

10

9

42

0

0

 

Total

198

0

0

 

Table 7: Summary of observations of the 100% v/v saturated solution test group

Day

Adults Surviving

Number of adults with eggs in brood pounch

Live young

Dead young

Unhatched Eggs

1

10

0

0

0

0

2

8

2

0

0

0

3

7

4

0

0

0

4

6

3

4

0

0

5

6

3

11

0

0

6

6

4

15

0

0

7

5

4

1

0

0

8

5

5

        31

0

0

 

Total

62

0

0

  

Table 8: Total cumulative production of live young

 

Nominal Concentration (% v/v saturated solution)

Day

1

2

3

4

5

6

7

8

Control

0

0

13

28

76

116

190

245

10

0

0

11

24

69

124

183

214

32

0

0

12

20

75

124

156

198

100

0

0

0

4

15

30

31

62

  

Table 9: Number of live young produced per adult (Non-cumulative) 

 

Mean Measured Concentration (mg/L as Test Material)

Day

1

2

3

4

5

6

7

8

Control

0

0

1

2

5

4

8

6

10

0

0

1

1

5

6

6

3

32

0

0

1

1

6

5

4

5

100

0

0

0

1

2

3

0

6

 

 

 

 

 

  

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Ceriodaphnia dubia to the test material resulted in significant mortalities at the test concentration of 100% v/v saturated solution resulting in 40% mortalities by Day 8. The 8-Day EL50 (immobilisation) value, based on nominal test concentrations, for the parental Ceriodaphnia generation (P1) was greater than 100% v/v saturated solution. The 8-Day EL50 (reproduction) based on nominal test concentrations was 78% v/v saturated solution.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 100% and 32% v/v saturated solution, respectively.
Executive summary:

The effect of the test material on the survival and reproduction of Ceriodaphnia dubia over an 8-Day period was investigated in a study which was conducted under GLP conditions and partly in accordance with the standardised guidelines OECD 211 and EPA OPPTS 850.1300.

As the test material is considered to be poorly soluble in water saturated solutions of test material were prepared for use in the study.

Based an acute toxicity to Daphnia magna study (Harlan Laboratories Ltd Project Number: 2702/0173), Ceriodaphnia dubia were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of test concentrations of 10, 32 and 100% v/v saturated solution for a period of 8 days. The test material was prepared by stirring an excess of the test material in reconstituted water (Elendt M4) water using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 25°C for 48 hours. After stirring, any undissolved test material was removed by filtration through a pre-conditioned 0.2 µm filter to produce the 100% v/v saturated solution. A series of dilutions was made from this saturated solution to prepare the remainder of the test series. The test solutions were renewed 3 times. The numbers of live and dead adult Ceriodaphnia and young daphnids (live and dead) were determined daily. The Ceriodaphnia were fed daily with an algal suspension and YAT (yeast, alfalfa, trout chow) combination.

The 8-Day EL50 (immobilisation) value based on the nominal test concentrations, for the parental Ceriodaphnia generation (P1) was estimated to be greater than 100% v/v saturated solution. The 8-Day EL50 (reproduction) value based on the nominal test concentrations was calculated to be 78% v/v saturated solution. The "Lowest Observed Effect Concentration" was considered to be 100% v/v saturated solution on the basis that at this test concentration significantly fewer live young (P < 0.05) were produced during the test compared to the control. The "No Observed Effect Concentration" was considered to be 32% v/v saturated solution on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P > 0.05) between the control and the 32% v/v saturated solution test group in terms of numbers of live young produced per adult by Day 8.

As it is not possible to ascertain the levels of test material in solution and the relevant effect levels it is not possible to derive an Environmental Reference Value using the results from the study. Furthermore, results from the study are not directly relevant to apply to classification and labelling for long term environmental toxicity as the study was only conducted over 8 days rather than 21 days as required by the standard test guideline.

The findings from this study are therefore not useful and are disregarded in favour of findings of a 21-day study (Lilliput, 2013) which are regarded as a more reliable indication of the chronic toxicity of the test material to freshwater invertebrates.

Description of key information

21-day NOEC = 100 mg/L (nominal loading), Daphnia magna, OECD 211, Lillicrap (2013)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

Two studies are available addressing the long term toxicity of the test material to freshwater invertebrates:

The key study, reported by Lillicrap (2013) has a Klimisch reliability rating of 1. It was conducted under GLP conditions and in accordance with the standardized guideline OECD 211 using an appropriate test medium for metallic substances.

During this study Daphnia magna, < 24 hours old, were exposed to a range of concentrations of test substance prepared from a water soluble fraction based on an initial loading of 100 mg/L. The test was performed at a temperature of 20 ± 2°C under semi-static test conditions with renewal of the test medium. The test duration was 21 days. Daphnia exposed concurrently in daphnia media only, served as the control. Mortality, immobility, time to first production of young and number of young produced and length after 21 days were recorded during the study.

There were no effects on the survival of the daphnids at any concentration of the test substance during the study, no statistically significant reduction in daphnia offspring production at any concentration tested when compared to the control, and there were no significant reductions in the length of the daphnids at any concentration of test substance investigated. Therefore, under the conditions of the study the 21-day No Observed Effect Concentration (NOEC) was determined to be 100 mg/L (based on nominal values).

The second study, reported by Goodband & Mullee (2010), has a number of identified deficiencies and scientific controversies and as such has been assigned a Klimisch reliability rating of 3. The findings of this study are therefore disregarded on the following grounds:

 

1)  The use of a chelating agent to test a metallic substances including the registered substance: The test medium - Elendt M4 used in this study is stipulated in the OECD 211 guideline as an inappropriate medium for testing metallic substances as it contains EDTA which is a chelating agent.

2)  The presence of EDTA should theoretically decrease the toxicity of metals in solution, including the metallic impurities as EDTA forms especially strong complexes with Mn(II), Cu(II),Fe(III),Pb(II) and Co(III)) to name but a few but this is not the case in this study – an unexplained phenomenon. The results seem to be the an extreme opposite with Mn showing an increased toxicity with no scientific explanation to this, hence the reliability of the methodology, the analytical process and the observations are questionable.

3)  LOQ -the levels of manganese measured in solution were below the limit of detection. Therefore, an environmental reference value (ERV) could not be obtained using results from this study. If the levels were well and truly below the detection limit, for a substance which is considered insoluble, the bioavailable concentration should have been low. At such low concentration and given the well known essentiality aspects of manganese it is unclear how this substance could have shown such apparent high levels of toxicity. 

4)  With no clear scientific rational and explanation behind the results of this study, one would wonder if the NOEC was derived or determined. Based on the above – the results of this study have been considered to be unreliable and have been considered a false positive. As it is not possible to ascertain the levels of test material in solution and the relevant effect levels it is not possible to derive an Environmental Reference Value using the results from the study. Therefore, the findings from this study are not useful and are disregarded in favour of findings of a 21-day study (Lilliput, 2013) which are regarded as a more reliable indication of the chronic toxicity of the test material to freshwater invertebrates.