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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: EEC Guideline XI/681/86, Draft 4: "Prolonged toxicity study with Daphnia magna: Effects on reproduction"
Principles of method if other than guideline:
For the statistical evaluation of the LOEC and NOEC Duncan's new multiple range test was used.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken in the 1st, the 2nd and the 3rd week of the test. For each concentration the freshly prepared test solution (unstocked) and the corresponding 48 h or 72 h old test solution (stocked with daphnids) were analyzed.
Vehicle:
no
Details on test solutions:
At the beginning of the test and before changing the test solutions, a stock solution of the test substance was freshly prepared. A defined amount of the substance was weighed out and suspended directly in the test medium by stirring (about 10 min at 22+/-1 °C). The nominal concentrations of the stock solutions were 100 mg/L. By dilution of the stock solutions with test medium, the nominal test concentrations were prepared.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna STRAUSS
Supplier: Institut National de Recherche Chimique Appliquée, France, 1978
Culture: The daphnids are cultured under standard conditions in the Laboratory for Experimental Ecology of BASF AG, Ludwigshafen
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Hardness:
Total hardness: 2.20 - 3.20 mmol/L
Test temperature:
19.9 - 21.7 °C
pH:
7.5-8.5
Dissolved oxygen:
Oxygen content: 8.0 - 9.6 mg/L
Nominal and measured concentrations:
Test concentrations (nominal): 100; 50.0; 25.0; 12.5; 6.25; 3.13 and 1.56 mg/L. (Dilution factor: 2)
Details on test conditions:
TEST PROCEDURE
- Semistatic procedure: Change of test solution three times a week.
- Test water: synthetic M4-medium
Alkalinity up to pH 4.3: 0.80 - 1.00 mmol/L
Molar ratio Ca:Mg: about 4 : 1
Conductivity: 550 - 650 µS/cm
- Number of animals/vessel: 1
- Number of replicates/concentration: 10
- Number of replicates/control: 10
- Age of the animals at the start of the test: 2 - 24 h
- Age of stock animals: 2 - 4 weeks
- Light: Artificial light, OSRAM L58 W31 warm white day : night-rhythm = 16 : 8 hours
- Removal of young from test beakers and counting: daily
- Check of the study and recording (mortality, hatching of young): daily
- Feeding: daily
- Measurement of temperature: Continuously in an extra vessel
- Measurement of pH and oxygen: At the start of the test and at each change of test solution in the 48
- or 72-hour-old solution in one parallel at each concentration
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
21 d
Dose descriptor:
other: LC0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Analytically found relative minimum and maximum concentrations of the test substance in the test solutions
at different times of the test (with and without daphnids). The values are given in percent of the nominal concentrations:


Time / Stocked with / Minimum / Maximum /
[h] / daphnids / [%] / [%] /
-----------------------------------------------------------
0 / no / 80.8 / 166.7 /
48/72 / no / 92.8 / 166.7 /
48/72 / yes / 95.2 / 173.1 /
-----------------------------------------------------------
M4-water caused a background value of about 1 mg/L "Trilon BD". Regarding to this and to the precision of the analytical method, most concentrations found corresponded to the expected values.


Validity criteria:

In the control:
- the mortality of parent animals up to the end of the test was <= 10 %.
- the mean number of living young per parent animal that survived the test was >= 60.
- the coefficient of variation for the mean number of living young per parent animal that survived the test was <= 25 %.
- The NOEC of the control substance 3,4-dichloroaniline was 2.5 ug/L, the LOEC was 10 ug/L and LC0 was > 10 ug/L.

The validity criteria were fulfilled by the test.


Control:
In the control the first young were observed at day 7. In the highest concentration tested, at which the daphnids produced young (100 mg/L), the first young were observed at day 13.
Conclusions:
Chronic toxicity to Daphnia resulted in a NOEC of 25 mg/L and a LOEC of 50 mg/L
Executive summary:

Chronic toxicity to Daphnia resulted in a NOEC of 25 mg/L and a LOEC of 50 mg/L

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Excluding the following: Ammonium, Nitrate-N, total phosphate and hardness measurements of HD water.
Analytical monitoring:
yes
Details on sampling:
Samples of all the test concentrations were taken on preparation (fresh) and after changing (used) of the test solution.
The volume of the samples of the new test concentrations was 10 mL. For sampling after changing the test solutions, 1 mL was taken from every replicate and pooled, these pooled samples were filtered over a 0.45 µm GHP Acrodisc filter to remove the algae. The samples were stored in a refrigerator until analysis.

To check the stability of the storage conditions, 3 control samples in test medium from the middle of the concentration range were stored under the same storage conditions as the samples taken at the start of the test. After finishing the test, these control samples were analyzed to check the stability, by comparing them to 3 samples freshly prepared at the time of analysis.

For the standard solutions and the test samples 1 mL of complexing solution was pipetted in a 5 mL syringe with a 0.2 µm filter at the end. Exactly 4 mL of standard solution or test sample was added and the syringe was closed with the piston. The samples of 500 mg/L test solution were diluted 10 times (0.4 mL sample was diluted with 3.6 mL HD water).
The syringe was shaken and allowed to stand for 15 minutes. After the reaction time samples were filtered into a HPLC vial and directly analyzed.
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions
To prepare the stock solutions for every water renewal, on average 1.0007 g (standard deviation: 0.0004 g) of test substance was weighed on an analytical balance then dissolved directly in 100 mL HD water with vitamins. The obtained preparations were agitated mechanically for approximately 1 to 2 hours in an attempt to completely dissolve the test substance. The pH of each stock solution was checked and found to be too low, therefore it was adjusted with 1M sodium hydroxide solution to a pH of 7.5 to 7.9.
A fresh stock solution was prepared for each solution change.

Preparation of the test solutions
Test solutions were prepared by further dilution of the stock solution with HD water with vitamins.
A geometric series of concentrations was used. The ratio between two consecutive concentrations was approximately 3.2.
Test vessels were filled directly from the test solution containers immediately after preparation. The solutions were renewed at least three times a week during the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: Dr .U. Noack-Laboratorien Käthe-Paulus- Str. 1, D-31157 Sarstedt, Germany) cultured in conformity with the relevant SOP. The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use).

Parent animals were fed a diet of 0.1 to 0.2 mg of carbon per daphnid per day, in the form of the algal strain Chlorella vulgaris. The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has previously been measured.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
min. 5.7; max. 6.7 mg CaCO3/L
Test temperature:
min. 20.8; max. 21.6 °C
pH:
min. 7.2; max. 8.7
Dissolved oxygen:
min. 4.4; max. 9.8 mg O2/L
Nominal and measured concentrations:
nominal: 4.7, 15.2, 48.8, 156 and 500 mg/L

The chemical analyses showed that the measured concentrations were within 80 to 120% o the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glas beaker of 50 mL
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution: three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Natural surface water was sampled from Heveadorp in the district of Renkum, The Netherlands, coordinates: 51º 58’N 5º48’E. This sample point has a historically low DOC and TSS concentration.
Heveadorp water (HD water) was frozen until use.

Sampling Date: 03-30-2010
Weather: Cloudy
Appearance: Clear, light yellow
pH: 7.9
Conductivity (µS/cm): 314
DOC 1 (mg/L): 2.612
TSS (mg/L): 3.6
Ammonium- N 2 (mg/L): 0.117
Nitrate- N 2 (mg/L): 5.10
Total Phosphate 2: Not detectable
Total hardness: 5.92 °dH, Ca 38.2, 3 mg/L / Mg Not detectable 2
1: Higher than the recommended maximum value of 2 mg/L
2: Not measured under GLP
3: Below the recommended minimum value of 140 mg/L

The DOC concentration was higher than recommended in the guideline. As MnNa2-EDTA does not adsorb onto organic matter it is not considered to have an impact on the outcome of the test. Water hardness was lower than recommended in the guideline.
But because the daphnids showed excellent performance in this test medium it is not considered to have an impact on the outcome of the test.

OECD recommended M4 media vitamins (9.4) were added to HD water to create the test medium. This was done in the normal manner as described for Elendt M4 medium. Test media was aerated prior to use with filtered air.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h
- Light intensity: 10.3 µmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Animals were checked for immobilisation of parent daphnids on at least 6 days per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other abnormal observations were also recorded.
At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 mm using a binocular microscope. Following measurement of body length, parent animals were placed in an oven at 105 °C overnight in groups and weighed (to constant dry weight) to calculate the mean individual weight per surviving parent animals per concentration.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
At first a definitive test was performed in enriched M4 medium. Due to elevated pH and mechanical problems this test was terminated. Measures were taken to keep a more constant pH and a second definitive test was performed in enriched M4 medium. This test was stopped as well, due to poor performance of the daphnids. After this, natural surface water with M4 vitamins was chosen as test medium (see also section 7.1).

The test concentrations were based on a 10-day range finding study in natural surface water. A range of 3 concentrations, namely 4.7, 48.8 and 500 mg/L were tested. Positive effects were seen at 4.7 and 48.8 mg/L. At 500 mg/L reproduction was significantly lower than in the control.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
156 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
reproduction
Remarks on result:
other: Very soft water
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
156 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
other: length
Remarks on result:
other: Very soft water
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
48.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
other: weight
Remarks on result:
other: Very soft water
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
365 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
397 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
198 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
348 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: length
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
91.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: length
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
156 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight
Details on results:
No mortality occurred in the control group over the test period.
The average number of juveniles per parent in the control was 111 after 21 days.

An EC50 based on survival of parent animals could not be determined because only two animals died during the test, one in 15.2 mg/L and one in 500 mg/L.

The first brood was released on day 8. One animal in the control and two in 156 mg/L had the first brood on day 7. In 48.8 and 500 mg/L one animal released the first brood only on day 12, one animal in 156 mg/L on day 9.
In 4.7 mg/L on one occasion one dead young was found in one replicate. In 15.2 mg/L on one occasion 4 dead young were found in one replicate. In 500 mg/L several dead young were found in several replicates.
On two occasions in 500 mg/L some aborted eggs were seen in 3 replicates.
In 500 mg/L the parent animals started to look pale on day 16 of the test.

A thin brown film was seen on the water surface in all test concentrations. In some test concentrations brown precipitate was also seen.
Results with reference substance (positive control):
A short-term reference test with potassium dichromate was performed in HD water with M4 vitamins. The results of this test were within the range given in the OECD 202 Guideline.
Reported statistics and error estimates:
The data on reproduction was tested for normality using Kolmogorov D test and found to be normally distributed. The data passed Bartlett’s tests for homogeneity of variance. Analysis of variance was performed on the number of living neonates per parent using the Dunnett’s test.
An EC10 and EC50 were determined applying probit analysis using Toxcalc v 5.0.
Length data were found to be normally distributed and displayed homogeneity of variance. For weight data normality could not be confirmed because animals were pooled in only two groups per test concentration to be able to weigh accurately. The data passed Bartlett’s tests for homogeneity of variance.

Number of juveniles per parent alive at end of the test at each test concentration, mean number of neonates per concentration and coefficient of variance.

 

Concentration (mg/L)

Rep no.

0

4.7

15.2

48.8

156

500

1

110

103

117

114

105

23

2

112

119

110

114

115

23

3

107

94

114

120

118

28

4

110

122

110

113

118

26

5

103

93

120

138

110

27

6

117

123

124

106

111

47

7

113

83

92

109

115

D

8

116

96

110

136

92

26

9

118

95

D

122

83

26

10

106

108

94

119

103

30

Mean

111

104

110

119

107

28

St. dev.

5

14

11

11

12

7

CV (%)

4

13

10

9

11

26

D = died during the test

Length of adult daphnids (mm)

Daphnia No.

Control

4.7 mg/L

15.2 mg/L

48.8 mg/L

156 mg/L

500 mg/L

1

4.7

4.9

4.9

4.5

4.7

4

2

4.9

4.9

4.6

4.7

4.8

4.1

3

4.8

4.9

4.5

4.9

4.8

4.2

4

4.7

5.0

4.4

4.7

4.7

4.3

5

4.9

4.4

4.9

5

4.9

4.4

6

4.8

5.0

4.9

4.5

4.9

4.1

7

5.2

4.8

4.8

4.6

4.6

-

8

4.6

4.8

4.7

4.4

4

3.8

9

4.8

4.9

-

4.6

4.4

3.9

10

4.7

4.7

4.8

4.8

4.9

4.3

Mean

4.8

4.8

4.7

4.7

4.7

4.1

Weight of adult daphnids

Conc.

replicate

No. of daphnids

Total weight (g)

Weight per daphnid (mg)

Control

I

5

0.0061

0.0012

II

5

0.0066

0.0013

4.7 mg/L

I

5

0.0065

0.0013

II

5

0.0055

0.0011

15.2 mg/L

I

5

0.0056

0.0011

II

4

0.0053

0.0013

48.8 mg/L

I

5

0.0063

0.0013

II

5

0.0060

0.0012

156 mg/L

I

5

0.0049

0.0010

II

5

0.0052

0.0010

500 mg/L

I

5

0.0027

0.0005

II

4

0.0021

0.0005

Validity criteria fulfilled:
yes
Conclusions:
The test was well conducted according to guideline and GLP (excluding Ammonium, Nitrate-N, total phosphate and hardness measurements of HD water).
The test substance does not show long-term toxicity to Daphnia magna. A 21d-EC10 and 21d-EC50 were determined as 91.9 mg/L and 397 mg/L, respectively.
Executive summary:

A prolonged toxicity test with Daphnia magna STRAUS - clone 5 was preformed in a semi-static test according to OECD Guideline No. 211, 3 October 2008 and EU guideline C.20 from Annex V of Directive 67/548/EEC (Gyimesi 2010, Rel. 1).

  

The nominal concentrations used in the study were as follows: 0, 4.7, 15.2, 48.8, 156 and 500 mg/L.

All concentrations given refer to the technical product as supplied by the sponsor.

 

Analytical determinations of the test solutions were made on 6 occasions during the test.

The concentrations were found to remain within 80 and 120% of the nominals for 4.7and 500 mg/L. As all concentrations measured are close to the nominals the effect data are based on the nominal test concentrations.

 

The validity criteria were respected:

No mortality occurred in the control group over the test period.

The average number of juveniles per parent in the control was111after 21 days.

  

Parental weight was found to be the most sensitive endpoint based on the EC10 values.

An EC10 and EC50 for parental weight were determined at 91.9 mg/L and 397 mg/L, respectively. The EC10and EC50 for reproduction were 198 mg/L and 365 mg/L, respectively.

Using Dunnett’s test, the Lowest Observed Effect Concentration (LOEC) was found to be 156 mg/L for parental weight. Based on these statistical results the NOEC is 48.8 mg/L.

The EC50 for parent survival could not be determined because only two animals died during the test.

Description of key information

Key value for chemical safety assessment

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

Additional information

There are no studies available on the long term toxicity of trisodium HEDTA to aquatic invertebrates, however the effects of disodium EDTA and manganese disodium EDTA were investigated in 21 d studies conducted withDaphnia magna. The results indicated a 21 d NOEC (reproduction) of 25 mg/L, expressed as Trilon BD (containing Na-EDTA) in hard water. Assuming a purity of 100%, this is equivalent to ca. 25.6 mg Na3-HEDTA (based on a molecular weight of 336.21 for Na2-EDTA and 344.20 for Na3-HEDTA). For MnNa2-EDTA, the 21 d NOEC were 48.8 (weight) or 156 (length and reproduction) mg/L in very soft water. Recalculated as Na3-HEDTA, this is equivalent to ca. 43.1 or 137.99 mg/L (based on a molecular weight of 389.13 for MnNa2-EDTA and 344.20 for Na3-HEDTA). The lowest value of 25.6 mg/L was retained for the CSA.