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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Study according to "Standard methods for the examination of water and wastewater (APHA-1975) using preferred species. Exposure is 72 hours instead of 48 hours as described in OECD202, however, this is not expected expect to affect the conclusion since the LC50 value after 72-hours of exposure is very high.
Qualifier:
according to guideline
Guideline:
other: Standard methods for the examination of water and wastewater. 14th ed., American Public Health Association, New York (1975)
Deviations:
not specified
Principles of method if other than guideline:
D. carinata collected from local ponds were acclimated to laboratory conditions for four days. Bioassays were performed according to American Public Health Association. Standard Methods for the Examination of Water and Wastewater. 1975. Experiments were run at 22.7°C and 30.8°C for 72-hours in 500 mL beakers using unchlorinated well water at pH 7.0. Dissolved oxygen was 7.6 ppm and total alkalinity was 150 ppm as CaCO3. Twenty animals were kept per container and six replicates were conducted for each test and control.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: No data
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
not specified
Details on test solutions:
No data
Test organisms (species):
other: Daphnia carinata (water flea)
Details on test organisms:
The zooplankton Daphnia carinata, Moina micrura and Cyclops viridis and larvae Chironomus and Dragon fly nymphs were collected from local ponds were acclimated to laboratory conditions for four days.

In addition, worm and molluscs were also collected from local ponds and tested.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
total alkalinity 150 ppm as CaCO3
Test temperature:
22.7°C and 30.8°C
pH:
7.0
Dissolved oxygen:
7.6 ppm
Salinity:
No data
Nominal and measured concentrations:
No data
Details on test conditions:
Twenty animals per container and six replicates for each test were conducted alongwith controls.
Unchlorinated tubewell water was used (pH 7.0. Dissolved oxygen 7.6 ppm, total alkalinity 150 ppm as CaCO3, ammonia nitrogen 0.08 ppm,
phosphate 0.05 ppm)
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 790 mg/L
Basis for effect:
mortality
Remarks on result:
other: Daphnia carinata, 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 825 mg/L
Basis for effect:
mortality
Remarks on result:
other: Daphnia carinata, 30.8°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 650 mg/L
Basis for effect:
mortality
Remarks on result:
other: Moina micrura, 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 600 mg/L
Basis for effect:
mortality
Remarks on result:
other: Moina micrura, 30.8°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2 260 mg/L
Basis for effect:
mortality
Remarks on result:
other: Cyclops viridis, 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2 350 mg/L
Basis for effect:
mortality
Remarks on result:
other: Cyclops viridis, 30.8°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 520 mg/L
Basis for effect:
mortality
Remarks on result:
other: Chironomus, 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 500 mg/L
Basis for effect:
mortality
Remarks on result:
other: Chironomus, 30.8°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 145 mg/L
Basis for effect:
mortality
Remarks on result:
other: Dragonfly nymph, 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 120 mg/L
Basis for effect:
mortality
Remarks on result:
other: Dragonfly nymph, 30.8°C
Details on results:
Number Daphnia carinata tested = 700.
The lowest LC50 value of all organisms tested was 1120 mg/L for Dragon fly nymph at 30.8°C.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
No data

The read-across rationale can be found in the category approach document attached in Section 13 of IUCLID and is fully incorporated in the CSR.

Validity criteria fulfilled:
not specified
Conclusions:
The LC50 value to Daphnia carinata after 72-hours of exposure at both 20.7°C and 30.8°C is very high (1790 and 1825 mg/L, respectively).
The lowest LC50 value of all organisms tested was 1120 mg/L for Dragon fly nymph at 30.8°C.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test groups
- Sampling method: 100 mL at t= 0 from the bulk test preparation; 100 mL at t=48 h from the pooled replicates
- Sample storage conditions before analysis: in the freezer
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Range-finding, initial and confirmatory tests:
- Method: A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations (RF: 0.1, 1.0 and 10% v/v saturated solution; confirmatory tests: 10, 18, 32 and 56% v/v saturated solution).
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Controls: test water without test item

Definitive test:
- Method: A nominal amount of test item (5500 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item. Chemical analysis of the saturated solution showed that a measured concentration of 396 mg/L was obtained. A series of dilutions was made from this saturated solution to give the required test concentrations of 10, 18, 32, 56 and 100 mg/L.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Controls: test water without test item
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture
- Age at study initiation: <24 h old
- Method of breeding: 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.
- Feeding during test: no

ACCLIMATION
- Acclimation period: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
21°C
pH:
7.1-7.6 (definite test)
Dissolved oxygen:
8.3-8.9 (definite test)
Nominal and measured concentrations:
Definite test
nominal: 10, 18, 32, 56 and 100 mg/L
measured concentrations:
- at t=0 h: 8.88, 16.3, 28.6, 49.6 and 89.5 mg/L
- at t=48 h: 8.73, 16.6, 29.1, 50.9 and 89.7 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel, material, size, fill volume: 150 mL glass beaker covered with a lid to reduce evaporation, 100 mL fill volume
- Aeration: not during test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium)
- Culture medium different from test medium: yes (culture medium: Elendt M7)
- Intervals of water quality measurement: 24 h (t=0, 24, 48)

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary
- Photoperiod: 16 h light and 8 h darkness with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilization or adverse reactions to exposure (at 24 and 48 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

* Range finding study
- Test concentrations:0.1, 1.0, 10 and 100 % of a saturated solution prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes - the loading rate was not high enough to achieve the desired test concentration of 100 mg/L (actual concentration was 75 mg/L in RF), so in the definitive test the loading rate was increased to 500 mg/L to assure for sufficient dissolved test item in test medium; 1 out of 10 daphnids was immobile in the RF at the highest tested concentration (i.e. 100 % of a saturated solution prepared at a loading rate of 100 mg/L, measured conc. of 75 mg/L)
Reference substance (positive control):
yes
Remarks:
potassium dichromate ((test performed in september 2015))
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 5% cumulative immobilitzation at 100 mg/L
Details on results:
- Behavioural abnormalities: after 24 hours, in the 18 and 32 mg/L test concentrations some daphnids were trapped at the surface
- Other biological observations: no
- Mortality/immobility of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.54 mg/L (95% C.I. 0.47-0.63 mg/L)
Reported statistics and error estimates:
An estimate of the EC50 values was given by inspection of the immobilization data.

Cumulative Immobilization Data and Observations in the Definitive Test

 

Nominal

Concentration

24 Hours

Cumulative Immobilized Daphnia

(Initial Population: 5 Per Replicate)

Observations

[mg/L]

R1

R2

R3

R4

total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

3 N 2 T

32

0

0

0

0

0

0

5 N

4 N 1 T

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

1

0

0

0

1

5

4 N

5 N

5 N

5 N

 

Nominal

Concentration

48 Hours

Cumulative Immobilized Daphnia

(Initial Population: 5 Per Replicate)

Observations

[mg/L]

R1

R2

R3

R4

total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

1

1

5

5 N

5 N

5 N

4 N

32

0

1

0

0

1

5

5 N

4 N

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

1

0

0

0

1

5

4 N

5 N

5 N

5 N

 

N = Normal

T = Trapped at the surface

 

 

Results for Test Samples in the Definitive Test

 

Time Point

Nominal Concentration of Test Item in Test Sample

Sample Preparation Factor

 

Determined Concentration of Test Item in Test Sample

% of Nominal Concentration

% relative to initial

 

cnom

F

c

 

 

[hours]

[mg/L]

 

[mg/L]

[%]

[%]

0

Control

1.02

<LOQ

-

 

 

10

1.02

8.88

89

 

 

18

2.04

16.3

91

 

 

32

3.06

28.6

90

 

 

56

5.10

49.6

89

 

 

100

10.2

89.5

90

 

48

Control

1.02

<LOQ

-

-

 

10

1.02

8.73

87

98

 

18

2.04

16.6

92

102

 

32

3.06

29.1

91

102

 

56

5.10

50.9

91

103

 

100

10.2

89.7

90

100

LOQ = Limit of Quantification

% relative to initial was calculated by the reviewer

Validity criteria fulfilled:
yes
Remarks:
(1. none of the control daphnids showed immobilization or other signs of disease or stress, 2. the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels)
Conclusions:
The 48-h EC50 of the test item to Daphnia magna was found to be in excess of 100 mg/L, based on nominal test concentrations.

Description of key information

Based on reliable studies performed with two analogue substances from the phosphate category, the 48h-EC50 of the substance is >100 mg/L. The read-across rationale can be found in the category approach document attached in IUCLID section 13 and the CSR.

    
    
    
    
    
    
    
    
    
    

study performed with single superphosphate an EC50 of 1790 mg/l at 20.7°C was determined. The read-across rationale can be found in the category approach document attached in Section 13 of IUCLID and is fully incorporated in the CSR

Based on reliable studies performed with two analogue substances from the phosphate

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

No reliable study with the substance is present, however two reliable studies with substances from the phosphate category are available.

In a reliable study with diammonium hydrogenorthophosphate (DAP) performed according to OECD 202 (in 2016), 20 daphnids per concentration were exposed for 48 hours in a static set-up at 21°C to the following range of nominal test concentrations: 10, 18, 32, 56 and 100 mg/L. Test concentrations were analytically verified (ICP-MS) and found to a) be in the range 87 -92% of nominal and b) remain stable (based on P-content) throughout the test period (98 -103% relative to initial). Therefore it is justified to base the endpoint on nominal test concentrations. Water quality parameters were maintained throughout the test. The acute toxicity of DAP to Daphnia magna, expressed as EC50, was exceeding the highest tested concentration, thus 48-h EC50 is >100 mg/L.This result (not acutely toxic to D. magna) is in line with data available for another analogue substance from the phosphate category. In a reliable study performed according to "Standard Methods for the Examination of Water and wastewater (APHA-1975)", Daphnia carinata collected from local ponds were exposed to single superphosphate for 72 hr. Although the recommended exposure time is 48 hr, the extended exposure period is not expected to have affected the study as the LC50 values are high. LC50 values after 72 -hours of exposure at both 20.7°C and 30.8°C are 1790 and 1825 mg/L, respectively.