Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 May 2010 to 26 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline (the deviation observed was not considered to have compromised the validity of the study and results); adequate coherence between data, comments and conclusions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
the temperature was below 13°C at T96 hours of the test
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
as cited above
Qualifier:
according to
Guideline:
other: OECD Series on testing and assessment, Guidance document on aquatic toxicity testing of difficult substances and mixtures, Number 23, December 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perchlorate d'Ammonium
- Substance type: monoconstituent
- Physical state: white powder
- Analytical purity: 99.9%
- Purity test date: 15 March 2010
- Lot/batch No.: A001/10
- Expiration date of the lot/batch: 31 January 2015
- Storage condition of test material: at room temperature and protected from humidity

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Sample frequency:
Samples were taken in the fresh solutions (i.e. at the start of the test and at each solution change), just before distribution in aquariums from the preparation containers.
Samples were also be taken in the old solutions (i.e. at each solution change and at the end of the test) in each aquarium directly.

Combined range finding and limit test:
Two samples of 5 mL were taken in the limit test solution (100 mg/L) and in the test solutions at 500 mg/L and 200 mg/L.
As the test item was not found to be toxic at 200 and 100 mg/L nominal, chemical analysis was undertaken to measure the concentration of the test item in this solution.

Samples preparation:
- Test solution (100 mg/L)
To achieve a target concentration of 0.8 mg/L of Perchlorate d’Ammonium, an aliquot (0.8 mL) of each test item solution was sampled into a 100-mL volumetric flask and each flask was completed to volume with diluent before injection.
Stability period/storage condition: 6 days of storage at -20°C.

- Test dosage forms (200 mg/L)
An aliquot (1 mL) of each test item solution was sampled under into a 100-mL volumetric flask and each flask was completed to volume with diluent. To achieve a target concentration of 0.8 mg/L of Perchlorate d’Ammonium, an additional dilution, 0.8 mL of previous solution with 1.2 mL of diluent, was carried out before injection.
Stability period/storage condition: 6 days of storage at -20°C.

From the aliquot of dosage form sampled (weighed accurately), the real volume of the aliquot analyzed was determined (using the density of each dosage form) and therefore the value of the first dilution factor was calculated.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

The stock solutions, for the combined range finding and limit test were prepared by dissolving the test item directly in test medium (see § "Conditions of housing").

The conditions of preparation of the stock solutions are reported in the following table:

Quantity of test item Volume of test water Concentration of the Duration of the agitation (mg) (mL) test item (mg/L) (minutes)

Combined range 6000 12000 500 5
finding and limit 2500 5000 500 5
test

After agitation, the stock solutions were used to prepare the test solutions.

Test solutions were prepared by further dilution of the stock solution with test medium to provide a geometric series of concentrations:
- 0.14, 1.4, 14, 100, 200 and 500 mg/L for the combined range finding and limit test.

The pH range considered as appropriate for a normal life of test organisms is circa 6 to 9.
The pH of all test solutions remained within the range 6.0 +/- 0.2 to 9.0 +/- 0.2 after preparation and there was therefore no adjustment of pH before incorporation of the animals.

Fishes were placed in test vessels which were gently filled with the test solutions after their preparation. The same vessels were used throughout the test. Solutions were changed every 24 hours over the 96-hour test period.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Pisciculture des godeliers, 27210 Le Torp, France
- Length at study initiation (length definition, mean, range and SD): from 40 to 60 mm long
- Feeding during test: none

ACCLIMATION
- Acclimation period: at least 12 days before the start of the test
- Acclimation conditions (same as test or not): same as test, except for aeration (dissolved oxygen concentration at or above 80% of the saturation value at the selected temperature, instead of at or above 60% during test)
- Type and amount of food: trout chow
- Feeding frequency: twice daily
- Health during acclimation (any mortality observed): no mortality was recorded

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:
between 10 to 250 mg/L as CaCO3
Test temperature:
between 13°C and 17°C and not ranging by more than ± 2°C
pH:
between 6 and 8.5 and did not vary by more than 1 unit in the control
Dissolved oxygen:
≥ 60% of the air saturation value at that temperature
Salinity:
Not measured
Nominal and measured concentrations:
Measured concentrations in the test solutions (100 and 200 mg/L) were within ± 20% of the corresponding nominal values throughout the test (Table A). Therefore the test item was stable under the experimental conditions and the study results are therefore based on nominal concentrations.
Nominal concentration of 100 mg/L corresponded to measured concentrations of 95.3 mg/L (0 hour), 89.2 mg/L (24 hours), 98.0 mg/L (48 hours), 92.4 mg/L (72 hours) and 94.2 mg/L (96 hours).
Nominal concentration of 200 mg/L corresponded to measured concentrations of 186 mg/L (0 hour), 180 mg/L (24 hours), 195 mg/L (48 hours), 168 mg/L (72 hours) and 195 mg/L (96 hours).
Details on test conditions:
TEST SYSTEM
- Aeration: dilution water was aerated prior to addition of the test item and forced aeration was used during the test in order to restore the dissolved oxygen concentration ≥ 60% of the air saturation value
- Renewal rate of test solution (frequency/flow rate): 24 hours after beginning of the test
- No. of organisms per vessel: 2 groups of 7 fishes (limit test) and 3 groups of 5 fishes and 2 groups of 7 fishes (range-finding test)
- Biomass loading rate: loading of 8.1 g/L during the study

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In all test vessels, animals were inspected for visible abnormalities and mortality at 3, 6, 24, 48, 72 and 96 hours.
At the end of the range finding (for the test solutions at 500 mg/L and 200 mg/L) and limit test, length of fish was measured for all surviving animals after euthanasia. Any animal found dead during the combined range finding and limit at the solutions of 500, 200 and 100 mg/L was also measured before to be removed.

TEST CONCENTRATIONS
- Range finding study: concentrations of 0.14, 1.4, 14, 200 and 500 mg/L
- Test concentrations: no definitive test was performed as no mortality was observed at 100 mg/L (limit test) and the measured concentrations of the test item were within +/- 20% of this nominal value throughout the test
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: sublethal effects
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: sublethal effects
Details on results:
Mortality:
In the limit test, all animals exposed to the limit concentration of 100 mg/L were alive at the end of the test.
In the range-finding test, all animals exposed to the highest test concentration of 500 mg/L were dead within 24 hours of the start of the test.
The mortality in the test solutions at 200 mg/L was 14% at the end of the test.
The mortality at 0.14, 1.4 and 14 mg/L was 0% the end of the test.

Sub-lethal effects:
Sub-lethal effects were observed at 200 and 500 mg/L nominal.
At 200 mg/L sub-lethal effects included abnormally high respiratory rate for all the fish from the T3 hours and a severe loss of equilibrium from T72 hours was also observed for the fish found dead at T96 hours.
At 500 mg/L sub-lethal effects included abnormally high respiratory rate before death.

Any other information on results incl. tables

Validity criteria:

Three in every four validity criteria of the study were respected for the test:

the mortality in the control not exceeded one fish at the end of the test,

.constant exposure conditions across all treatments and control,

.measured test concentration was at least 80% of the nominal concentration, and test concentrations are kept within ± 20% of the mean measured concentration during the course of the test,

.the dissolved oxygen concentration remained ≥ 60% of the air saturation value throughout the test in the test solution except for the test solutions at 100 mg/L and 200 mg/L.

 

However no mortality occurred in the limit test concentration (100 mg/L) and dissolved oxygen concentration dropped only at 54.7 and 54.4 at T23.9 hours for the test solutions at 100 and 200 mg/L respectively and 56.1 and 53.0 at T 47.9 hours for the test solutions at 100 and 200 mg/L respectively. Furthermore no mortality occurred during the period between T0 hours toT47.9 hours when dissolved oxygen concentration was below 60%.

Consequently, as all the others validity criteria were met and as dissolved oxygen concentration did not impact the test, the test was considered valid. 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a 96-hours semi-static acute toxicity test with Oncorhynchus mykis, the LC50 of the test item Perchlorate d’ammonium was > 200 mg/L.
The NOEC was 100 mg/L.
Executive summary:

Methods

The study included a limit test and a range-finding test (performed simultaneously).

As the test was performed under semi-static, the solutions were changed after 24, 48 and 72 hours.

Four groups of seven fishes (0, 100, 200 and 500 mg/L nominal) and three groups of five fishes (0.14, 1.4 and 14 mg/L nominal) were exposed to the test item dissolved in test water for 96 hours.

Mortality and sub-lethal effects at each concentration were recorded at 0, 3, 6, 24, 48, 72 and 96 hours.

 

Chemical analysis were undertaken daily to measure the actual concentrationof the test item in the solution prepared at 100 and 200 mg/L nominal.

Results

Validity criteria:

Three in every four validity criteria of the study were respected for the test:

.           the mortality in the control not exceeded one fish at the end of the test,

.           constant exposure conditions across all treatments and control,

.           measured test concentration was at least 80% of the nominal concentration, and test concentrations are kept within ± 20% of the mean measured concentration during the course of the test,

.           the dissolved oxygen concentration remained ≥ 60% of the air saturation value throughout the test in the test solution except for the test solutions at 100 mg/L and 200 mg/L.

 

However no mortality occurred in the limit test concentration (100 mg/L) and dissolved oxygen concentration dropped only at 54.7 and 54.4 at T23.9 hours for the test solutions at 100 and 200 mg/L respectively and 56.1 and 53.0 at T47.9 hours for the test solutions at 100 and 200 mg/L respectively. Furthermore no mortality occurred during the period between T0 hours to T47.9 hours when dissolved oxygen concentration was below 60%.

Consequently, as all the others validity criteria were met and as dissolved oxygen concentration did not impact the test, the test was considered valid. 

 

Chemical analysis:

Measured concentrations in the test solutions(100 and 200 mg/L) were within ± 20% of the corresponding nominal values throughout the test.Therefore the test item was stable under our experimental conditions and the study results can be based on nominal concentrations.

Mortality:

In the limit test, all animals exposed to the limit concentration of 100 mg/L were alive at the end of the test.

In the range-finding test, all animals exposed to the highest test concentration of 500 mg/L were dead within 24 hours of the start of the test.

The mortality in the test solutions at 200 mg/L was 14% at the end of the test.

The mortality at 0.14, 1.4 and 14 mg/L was 0% the end of the test.

 

Sub-lethal effects:

Sub-lethal effects were observed at 200 and 500 mg/L.

At 200 mg/L sub-lethal effects included abnormally high respiratory rate for all the fish from the T3 hours and a severe loss of equilibrium from T72 hours was also observed for the fish found dead at T96 hours

At 500 mg/L sub-lethal effects included abnormally high respiratory rate before death.

 

The results and conclusions of the combined range finding and limit tests are summarized in the following tables:

Group

Control

Perchlorate d’ammonium

Concentration
(mg/Lnominal)

0

0.14

1.4

14

100

200

500

 

 

 

Effects

Mortality (%) - 24 h

0

0

0

0

0

0

0

Mortality (%) - 48 h

0

0

0

0

0

0

100

Mortality (%) - 72 h

0

0

0

0

0

0

100

Mortality (%) - 96 h

0

0

0

0

0

14

100

 

 

 

 

 

 

 

 

Sub-lethal effects (1)

N

N

N

N

N

Y

Y

(1) N: none; Y: observed

 

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

72 hours

96 hours

LC100

500

500

500

500

LC50

>200

>200

>200

>200

LC0

200

200

200

100

LOEC

200

200

200

200

NOEC

100

100

100

100

(1) LC100: lowest concentration tested with 100% mortality; LC50: Median (50%) Lethal Concentration (numbers in brackets correspond to the 95% confidence limits); LC0: highest concentration tested without mortality; NOEC:No Observed Effect Concentration; LOEC: Low Observed Effect Concentration

 

As no mortality was observed at 100 mg/L and the measured concentrations of the test item werewithin ± 20% of this nominal value throughout the test, the study was considered complete.

Conclusion

In a 96-hours semi-static acute toxicity test with Oncorhynchus mykis, the LC50 of the test item Perchlorate d’ammonium was > 200 mg/L.

The NOEC was 100 mg/L.