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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:
weight of evidence
Study period:
09 November 2015 - 11 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 202 and in compliance with GLP, without deviations that influence the quality of the results.
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, inital 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 (definitive test)
Dissolved oxygen:
8.3-8.9 (definitive test)
Nominal and measured concentrations:
Definitive 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
measured concentrations 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)
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

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.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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, we do not expect this 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 (migrated information):
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:
Daphnia carinata were collected from local ponds were acclimated to laboratory conditions for four days.

In addition, other zooplankton (Moina micrura, Cyclops viridis), worm, larvae 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:
LC0
Effect conc.:
68 mg/L
Basis for effect:
mortality
Remarks on result:
other: 22.7°C
Duration:
72 h
Dose descriptor:
other: LC5
Effect conc.:
80 mg/L
Basis for effect:
mortality
Remarks on result:
other: 22.7°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 790 mg/L
Basis for effect:
mortality
Remarks on result:
other: 22.7°C
Duration:
72 h
Dose descriptor:
other: LC95
Effect conc.:
5 115 mg/L
Basis for effect:
mortality
Remarks on result:
other: 22.7°C
Duration:
72 h
Dose descriptor:
LC100
Effect conc.:
5 800 mg/L
Basis for effect:
mortality
Remarks on result:
other: 22.7°C
Duration:
72 h
Dose descriptor:
LC0
Effect conc.:
75 mg/L
Basis for effect:
mortality
Remarks on result:
other: 30.8°C
Duration:
72 h
Dose descriptor:
other: LC5
Effect conc.:
82 mg/L
Basis for effect:
mortality
Remarks on result:
other: 30.8°C
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1 825 mg/L
Basis for effect:
mortality
Remarks on result:
other: 30.8°C
Duration:
72 h
Dose descriptor:
other: LC95
Effect conc.:
5 325 mg/L
Basis for effect:
mortality
Remarks on result:
other: 30.8°C
Duration:
72 h
Dose descriptor:
LC100
Effect conc.:
5 780 mg/L
Basis for effect:
mortality
Remarks on result:
other: 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
Validity criteria fulfilled:
not specified
Conclusions:
LC50 value after 72-hours of exposure at both 22.7°C and 30.8°C is very high (1790 and 1825 mg/L, respectively).

Description of key information

No reliable study with triple superphosphate is available. Based on a reliable study with diammonium hydrogenorthophosphat, the EC50 in Daphnia magna is determined to be >100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

No reliable study with triple superphosphate is available. Therefore, read-across from structural analogous substances is conducted. For details on the read-across approach please refer to the analogue justification provided in IUCLID section 13. Since the read-across is conducted with two closely related but different source substances a weight of evidence approach is applied and the most sensitive effect value is considered for deriving classification and conducting the hazard assessment, i.e. a worst-case scenario is followed.

The short-term toxicity of diammonium hydrogenorthophosphate (DAP) to Daphnia magna was tested in a guideline study following OECD 202 in 2016. Daphnids were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg DAP/L for 48 hours. Mortality or symptoms of intoxication were not observed. Thus the reported EC50 (48 h) is >100 mg/L (nominal).

The result is supported by a reliable study on the toxicity of single superphosphate (SSP) 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 it is not expected to have affected the outcome of the study as the LC50 values are high. LC50 values after 72-hours of exposure at both 22.7 °C and 30.8 °C are 1790 and 1825 mg/L, respectively.