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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 November - 16 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test groups
- Sampling method: direct retrieval from the test vessel (200 mL)
- 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)
- Test water: dechlorinated laboratory tap water

- Method: A nominal amount of test item (2200 mg) was dissolved, in duplicate, in 22 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 as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 μm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) prior to pooling to give the 100 mg/L test concentration. A series of dilutions was made from this test solution to give further test concentrations of 6.25, 12.5, 25 and 50 mg/L.
Each solution was mixed with a flat bladed stirrer for approximately 1 minute to ensure adequate mixing and homogeneity.

- Controls: test water without test item
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation: >4 weeks old
- Length at study initiation (mean, range and SD): 5.2 cm (SD = 0.20)
- Weight at study initiation (mean and range, SD): 1.2 g (SD = 0.16)
- Method of breeding: not reported
- Feeding during test: no

ACCLIMATION
- Acclimation period: 9 days
- Acclimation conditions: same as test
- Type and amount of food: commercial trout pellets
- Feeding frequency: not reported; feeding was discontinued approximately 25.5 hours prior to the start of the definitive test
- Health during acclimation (any mortality observed): no abnormalities reported, no mortality in the 7 days prior to the start of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
approx. 140 mg CaCO3/L
Test temperature:
13-15°C
pH:
7.5-8.1
Dissolved oxygen:
fresh solutions: 9.7- 10.5 mg O2/L
used solutions: 8.9- 10.0 mg O2/L
Nominal and measured concentrations:
nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L (based on range-finding test at 100 mg/L)
measured concentrations:
* fresh solutions - 5.68-6.08 mg/L, 11.3-12.6 mg/L, 25.9-26.2 mg/L, 52.4-52.9 mg/L and 99.2-101 mg/L
* old (used) solutions - 6.11-6.41 mg/L, 12.5-12.6 mg/L, 25.4-26.4 mg/L, 53.0-53.1 mg/L and 101-103 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25-30 L glass vessel
- Type: loosely closed (covered to reduce evaporation)
- Fill volume: 20 L
- Aeration: yes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.41 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality (after 3, 6, 24, 48, 72 and 96 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentration: 100 mg/L
- Results used to determine the conditions for the definitive study: yes (After approximately 46 hours exposure 1 out of 3 fish at 100 mg/L was observed to have a loss of equilibrium.)
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: at 50 and 100 mg/L fish were sitting at the bottom and showed increased pigmentation (see any further information on results incl. tables)
- Other biological observations: after approximately 72 hours of exposure 2 out of 7 fish at 100 mg/L were observed to be moribund
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no; the test item preparations were observed to be clear colorless solutions throughout the test
Reported statistics and error estimates:
The LC50 value at 96 hours was calculated by Probit analysis using Linear Maximum-Likelihood regression.
The NOEC at 96 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction.
All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Sub-lethal Effects of Exposure in the Definitive Test

 

Nominal Concentration (mg/L)

Sub-lethal Effects

Time (Hours)

3

6

24

48

72

96

Control

No abnormalities detected

7

7

7

7

7

7

6.25

No abnormalities detected

7

7

7

7

7

7

12.5

No abnormalities detected

7

7

7

7

7

7

25

No abnormalities detected

7

7

7

7

7

7

50

No abnormalities detected

7

7

7

-

-

-

Increased pigmentation

-

-

-

7

7

7

100

No abnormalities detected

7

7

6

-

-

-

Sitting at the bottom

-

-

1

-

-

-

Increased pigmentation

-

-

-

7

5

5

Moribund

-

-

-

-

2*

-

* Observation of sub-lethal effects recorded at approximately 72 hours, 2 fish from 100% v/v saturated solution found to be moribund, classed as mortality at 72 hours

Results for Test Samples (definite test)

 

Time Point

Nominal Concentration

 

 

Sample Preparation Factor

 

Determined Concentration of Test Item in Test Sample

% of Nominal Concentration

% relative to fresh (initial)

 

cnom

F

c

 

 

[hours]

[mg/L]

 

[mg/L]

[%]

[%]

0

Control

1.02

<LOQ

-

 

(fresh)

6.25

1.02

6.08

97

 

 

12.5

2.04

12.6

101

 

 

25

4.08

26.2

105

 

 

50

10.2

52.4

105

 

 

100

10.2

99.2

99

 

24

Control

1.02

<LOQ

-

-

(old)

6.25

1.02

6.11

98

100

 

12.5

2.04

12.6

101

100

 

25

4.08

25.4

102

97

 

50

10.2

53.0

106

101

 

100

10.2

101

101

102

72

Control

1.02

<LOQ

-

 

(fresh)

6.25

1.02

5.68

91

 

 

12.5

2.04

11.3

90

 

 

25

4.08

25.9

104

 

 

50

10.2

52.9

106

 

 

100

10.2

101

101

 

96

Control

1.02

<LOQ

-

-

(old)

6.25

1.02

6.41

103

113

 

12.5

2.04

12.5

100

111

 

25

4.08

26.4

106

102

 

50

10.2

53.1

106

100

 

100

10.2

103

103

102

LOQ = Limit of Quantification

% relative to initial was calculated by the reviewer

Validity criteria fulfilled:
yes
Remarks:
1) none of the control fish died or showed signs of stress during the test; 2) the oxygen concentration at the end of the test was ≥60% of ASV (6.2 mg O2/L) in the control and test vessels
Conclusions:
The 96-h LC50 of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) was found to be in excess of 100 mg/L, based on nominal test concentrations.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 24, 2000 - November 30, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg/L
- Sampling method: Water samples were collected at 0 (test initiation) and 96 hours (test termination) of the definitive toxicity test.
Control and test substance-fortified samples were also analyzed at each sample point.
At each sampling point, 6-mL samples were collected from each replicate of the control or exposure treatments and composited in culture tubes.
Samples were collected subsurface from the approximate center of each exposure chamber. At termination, an additional 500-mL control
composite sample was collected by combining approximately 250 mL of each control replicate in a 1-L beaker for preparation of quality control
fortification samples (QC spikes). Two QC spikes were prepared at each sample point at concentrations ranging from 3.13 to 106 mg MAP/L.
The low and high QC spikes were prepared by diluting MAP with dilution water at test initiation and with the control composite sample
at test termination. Samples were diluted, if necessary, with either dilution water (at initiation) or control composite sample (at termination)
so that the expected analyte concentrations were within the range of the calibration curve (1.00 to 20.0 mg total phosphate/L).
Vehicle:
no
Details on test solutions:
no data
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: obtained as fertilized eggs from Troutlodge, Inc., Sumner, WA, USA
- Age at study initiation (mean and range, SD): approximately five months
- Length at study initiation (length definition, mean, range and SD): 45 to 60 mm total length (mean and standard deviation = 53 ± 5.5 mm)
- Weight at study initiation (mean and range, SD): 0.662 to 1.768 g wet weight (mean and standard deviation = 1.226 ± 0.407 g)
- Method of breeding: Following hatch, fish were fed salmon starter and/or brine shrimp nauplii daily during the holding period.
Three days prior to test initiation, approximately 230 fish were removed from the holding tank and placed in acclimation.
- Feeding during test: fish were not fed during the test.


ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions (same as test or not):Acclimation water temperature ranged from 15.2 to 15.9°C.
- Type and amount of food: animals were not fed during acclimation
- Health during acclimation (any mortality observed): no mortality and no diseases observed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No data.
Hardness:
160 mg/L as CaCO3 at initiation
Test temperature:
15 ± 1°C
pH:
7.10 - 7.80
Dissolved oxygen:
Equal to or greater than 8.54 mg/L (equal to or greater than 88% of saturation) at initiation of the definitive test.
Levels of some of the test solutions were < 60% of saturation on study days 2 and 3, but did not fall below 5.63 mg/L in any chamber.
From study day 3 through termination, all solutions were gently aerated.
Levels were equal to or greater than 7.42 mg/L (76% of saturation) at termination.
Salinity:
Conductivity and temperatures were determined (see 'Test temperature' and 'Details on test conditions')
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25.0, 50.0, and 100 mg/L
Measured concentrations: 5.72, 11.9, 23.5, 46.6, and 85.9 mg/L (mean of t=0 and 96 h; 86 to 95% of nominal concentration)
The concentration of total phosphate in the control samples were detected at less than MDL (0.389 mg total phosphate/L or 0.598 mg MAP/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 5-gallon glass jars containing 15 L of control or test solution and covered with clear plastic petri dish
covers. The glass jars had measurements of approximately 47 cm (height) by 26 cm (inside-diameter) with 29 cm solution depth.
- Aeration: From study day 3 through termination, all solutions were gently aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: Instantaneous loading was calculated to be 0.41 g of fish tissue per liter of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a moderately hard laboratory freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis. These waters were blended to yield a total hardness of 130 to 160 mg/L as CaCO3.
The water was filtered prior to use.
- Alkalinity: 150 mg/L as CaCO3
- Conductivity: 304 uS/cm
- Total hardness: 160 mg/L as CaCO3
- Ammonia: Control samples, measured as total ammonia, were determined to be 0.062 and <0.012 mg/L on day 0 (test initiation)
and 0.28 and 0.30 mg/L on day 4 (test termination). Treatment samples, measured as total ammonia, increased proportionately with test
substance concentration and ranged from 1.2 to 18 mg/L on day 0 and from 0.66 to 18 mg/L on day 4.
Control samples, measured as un-ionized ammonia, were determined to be 0.0011 and <0.00023 mg/L on day 0 and both replicates
were 0.0036 mg/L on day 4. Treatment samples, measured as un-ionized ammonia, increased proportionately with test substance concentration and
ranged from 0.019 to 0.065 mg/L on day 0 and from 0.0069 to 0.16 mg/L on day 4.
- Intervals of water quality measurement: Temperature, dissolved oxygen, and pH were measured initially (prior to addition of fish) and daily
thereafter during the conduct of the test.In addition, total hardness, total alkalinity, and conductivity were measured at initiation of the definitive test.
All control and treatment solutions were analyzed for both total and un-ionized ammonia concentrations at initiation and termination of the test.


OTHER TEST CONDITIONS
- Photoperiod: Fluorescent lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods.
- Light intensity: 439 - 473 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and sublethal responses at test initiation and once every 24 ± 1 hours thereafter for the remainder of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: nominal total product test concentrations of 0.0 (control), 0.1, 1.0, 10, and 100 mg/L. Five fish were tested per
treatment.
- Results used to determine the conditions for the definitive study: No mortality or sublethal effects were observed in the control or any treatment
level following 96-hours of exposure.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
mortality
Remarks on result:
other: Nominal concentration confirmed by chemical analysis (>80% recovery)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Basis for effect:
other: mortality and sublethal effects
Remarks on result:
other: Nominal concentration confirmed by chemical analysis (>80% recovery)
Details on results:
No lethal or sublethal effects were observed at the highest concentration tested.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Statistical analysis of the concentration versus effect data (mortality) was not performed due to the absence of mortality during the definitive test.
The slope of the 96-hour dose-response line (log concentration vs. probit) could not be calculated due to the absence of fish mortality.

none

Validity criteria fulfilled:
yes
Conclusions:
Based on mean calculated total product test concentrations of Monoammonium Phosphate in test water, the nominal 96-hour LC50 value was determined to be >100 mg/L , the highest treatment concentration of the definitive test. The nominal 96-hour NOEC was 100 mg/L, based on
the absence of any mortality and sublethal effects at this and all lower treatment concentrations. Nominal concentrations were confirmed by chemical analysis (>80% recovery). The slope of the 96-hour dose-response line could not be calculated due to the absence of mortality.

Description of key information

No reliable study with single superphosphate is present. Based on reliable studies with diammonium hydrogenorthophosphate (DAP) and ammonium dihydrogenorthophosphate (MAP), the 96h-LC50 of the substance is > 100 mg/L. 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.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

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

In a reliable study with diammonium hydrogenorthophosphate (DAP) performed according to OECD 203 in 2016, 7 rainbow trout (Oncorhynchus mykiss) per concentration were exposed for 96 hours in a semi-static set-up (24 h renewal interval) at 13 -15°C to the following range of nominal test concentrations: 6.25, 12.5, 25, 50 and 100 mg/L. Test concentrations were analytically verified (ICP-MS) and found to a) be in the range 90 -106% of nominal and b) remain stable (based on P-content) throughout the 24 h renewal interval (97 -113% 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 rainbow trout, expressed as LC50, was exceeding the highest tested concentration, thus 96 -h LC50 is >100 mg/L.

In a reliable OECD 203 guideline study, rainbow trout were exposed for 96 hr under static conditions to 6.25, 12.5, 25.0, 50.0, and 100 mg/L ammonium dihydrogenorthophosphate (MAP). Measured concentrations were determined to be 5.72, 11.9, 23.5, 46.6 and 85.9 mg/L (mean of t=0 and 96 h; 86 to 95% of nominal concentration). Based on mean calculated total substance concentrations in test water, the nominal 96-hour LC50 value was determined to be >100 mg/L, the highest treatment concentration of the definitive test. The nominal 96-hour NOEC was ≥100mg/L, based on the absence of any mortality and sublethal effects at this and all lower treatment concentrations. Nominal concentrations were confirmed by chemical analysis (>80% recovery). The slope of the 96-hour dose-response line could not be calculated due to the absence of mortality.