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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 May 2010 to 20 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline (observed deviations were not considered to have compromised the validity or integrity of the study); 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 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the temperature varied by more than 2°C and the pH varied by more than 1.5 units in the control, during the test
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
as 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
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: January 2015
- Storage condition of test material: at room temperature and protected from humidity

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Two samples of 5 mL were taken from each test solution except for the control before the start of the test, just before the distribution to the test vessels. Samples were taken from each replicate with algae each 24 hours (T24, T48 and T72 hours) and then pooled in order to obtain two samples of 5 mL.
Two samples of 5 mL were taken at T24, T48 and T72 hours from the solutions which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test.

Chemical analysis was only performed on samples taken at the beginning T0 hour, at T24 hours and the end (T72 hours) of the test since measured concentration at T72 hours were within +/- 20% of the nominal values (and the initial values).

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST AND REFERENCE SOLUTION
The stock solutions, for the combined range-finding and limit test and definitive test, were prepared by dissolving the test item directly in OECD medium.
After agitation, the stock solutions were used to prepare the test solutions.

Test solutions were prepared by further dilution of the stock solution with OECD medium to provide a geometric series of concentrations:
- 0.1, 1, 10, 100, 200 mg/L and 500 mg/L for the combined range finding and limit test,
- 50, 88.9, 158.1, 281.2 and 500 mg/L for the definitive 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 addition of the algae.

Glass test vessels (250 mL Erlenmeyer flasks) were filled directly from the test solution containers after preparation and algae were then added to these vessels.
Test solutions remained unchanged throughout the study.

For the highest concentration test of the combined range-finding and limit test and definitive tests, a further group of vessels was prepared for chemical analysis by adding test solutions (except for the control) but no algae.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll PA37 1QA, Scotland
- Method of cultivation: the algae are cultured at CIT, under sterile conditions and maintained at exponential growth rate.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not):
. Culture medium: LC medium (reconstituted water prepared using deionized water with a conductivity less than 10 µS/cm). The LC medium after autoclaving has a pH of 7.5 ± 0.3.
. Culture period: algae were cultivated for 7 days before harvesting for use in a new culture. Each weekly culture was prepared under sterile conditions using autoclaved medium while week-old cell cultures were checked for contamination before being transferred to fresh medium.
. Temperature: between 21°C and 24°C in water. Temperature was checked daily.
. Illumination: 24 hours per day. Constant illumination was provided by white type fluorescent lights in the spectral range 40 to 700 nm with a light intensity of approximately 2000 lux.
. Aeration: the cultures were constantly aerated with air filtered using a 0.22 µm porosity filter to maximize the CO2 availability (and thereby the cell growth) while maintaining the sterility.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
During the combined range finding and limit test: 35.7 mg/L as Ca CO3
During the definitive test: 32.1 mg/L as Ca CO3
Test temperature:
During the combined range finding and limit test: between 22.1°C and 27.9°C
During the definitive test: between 23.8°C and 27.5°C
pH:
During the combined range finding and limit test: between 7.49 and 7.86 (between 8.07 and 9.99 for the control)
During the definitive test: between 7.51 and 9.30 (between 8.05 and 10.15 for the control)
Dissolved oxygen:
Not measured
Salinity:
Not measured
Nominal and measured concentrations:
Nominal concentrations of 50, 88.9, 158, 281 and 500 mg/L, corresponding to measured concentrations of:
- 49, 86.6, 158.9, 297.8 and 518 mg/L at T0h
- 48.5, 79.1, 132.1, 271.4 and 487 at T24h (without algae), 40.6, 85.7, 160, 256 and 447 at T24h (with algae)
- 21.8, 80.8, 145.7, 287.3 and 509 at T72h (without algae), 23.1, 92.8, 121.8, 278.1 and 488 at T72h (with algae)
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (see below)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 505 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
189.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
245.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
42.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

Growth inhibition (range-finding and limit test):

. Highest test concentration (500 mg/L)

Inhibitions of the average specific growth rate and yield at 500 mg/L nominal were 16.2% and 60.2%, respectively, relative to the control, after 72 hours.

. Range-finding and limit test

Average specific growth rate inhibitions at 0.1, 1, 10, 100 and 200 mg/L nominal were -1.2, 5.4, 1.3, 9.4 and 14.1%, respectively, relative to the control, after 72 hours.

Yield inhibitions at 0.1, 1, 10, 100 and 200 mg/L nominal were -7.0, 26.6, 7.1, 41.5 and 55.4%, respectively, relative to the control, after 72 hours.

Growth inhibition (definitive test):

The 24, 48 and 72 -hour ErC(x)s and the 72-hour EyCx were estimated from the % inhibition of average specific growth rate (ErC(x)s) and yield (EyCx).

The results and conclusions of the definitive test are summarized in the following tables: 

Group

Control

Perchlorate d’ammonium

Nominal concentration (mg/L)

 

0

50

88.9

158.1

281.2

500

 

 

 

Geometric mean of measured concentration (mg/L)

0 hour

ND

49.0

86.6

159

298

518

Geometric mean 0-24 h

ND

48.8

82.8

144.9

284.3

503

Geometric mean 0-72 h

ND

37.3

82.1

145.2

285.3

505

 

Inhibition (%)

Growth rate - 24 h

NA

-1.8

0.6

-1.3

4.1

4.8

Growth rate - 48 h

NA

-1.4

5.3

7.6

12.6

17.4

Growth rate - 72 h

NA

0.2

6.9

7.7

16.8

17.6

Yield - 72 h

NA

1.0

32.1

35.0

61.0

62.5

NA: not applicable, ND: not determined

 

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

72 hours

Growth rate

 

ErC50

> 503

> 503

> 505

ErC10

ND

ND

189.2

(126.6 – 266.8)

Yield

 

EyC50

ND

ND

245.6
(205.1 - 294.8 )

EyC10

ND

ND

42.2

(28.4 - 56.0)

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ECx is the test concentration where effect is reduced by x%.

The 24, 48-hour ErC50s were not calculated since the inhibition percentage of the average specific growth rate at the highest concentration (500 mg/L) was lower than 50% at the corresponding time (4.8 and 17.4 %, respectively).

The 72-hours ErC50 was calculated in the same time that the ErC10 but was not taking in account in the results and the conclusion of this report because this result was not considered as relevant since the inhibition percentage of the average specific growth rate at the highest concentration (500 mg/L) was lower than 50% at this time (17.6%).

The 72-hours ErC10 was calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

The 72-hour EyC50 and EyC10 were calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 of Perchlorate d’ammonium was > 505 mg/L based on average specific growth rate (ErC50) and 245.6 mg/L based on yield (EyC50).
Executive summary:

Methods

A preliminary test (combined range-finding and limit test) was performed to determine the concentrations to be tested in the definitive test.

Combined range-finding and limit test

. Limit test:

Three replicate algal suspensions with an initial dilution of 1 x 104 cells/mL were exposed to a nominal concentration of 100 mg/L and a further group of six replicate algal suspensions without test item was used as the control. The growth of the cell culture in each flask was monitored by counting the number of cells at 24, 48 and 72 hours.

. Range-finding test:

Five groups of two replicate algal suspensions were exposed to nominal concentrations of 0.1, 1, 10, 200 and 500 mg/L of the test item under the same conditions of light and temperature.

 

Definitive test

In the definitive test, algal suspensions were exposed to the nominal concentrations of 0, 50, 88.9, 158.1, 281.2 and 500 mg/L for 72 hours.

Three replicate algal suspensions were exposed to each test item concentration and six replicates to dilution water only (control) for 72 hours. Cell growth data were recorded at 24, 48 and 72 hours.

Samples were taken daily to measure the actual concentration of the test item in all solutions, except for the control, during the exposure period.

Results

All validity criteria of the study were respected.

All measured concentrations in the definitive test solutions with and without algae were within ± 20% of the corresponding nominal values throughout the test, except for the test solutions at 50 mg/L at T72 hours (with and without algae) and at 158 mg/L at T72 hours (with algae).

The results of T0 hours are presented as indicative values because samples were analyzed after 24 hours of storage at since no stability was demonstrated for storage 24 hours at.

Nevertheless, as the stability study showed that the test item was stable 24 hours under the test conditions of the test, it was considered that the values obtained with the samples taken at T24 hours were representatives to the values of T0 hours. Furthermore results (indicative) of T0 hours are also similar to the nominal values.

Consequently, the study results are based on geometric means of measured concentrations in test solutions without algae during the corresponding exposure period, i.e.24, 48 or 72 hours.

The 24, 48 and 72 -hour ErC(x)s and the 72-hour EyCx were estimated from the % inhibition of average specific growth rate (ErC(x)s) and yield (EyCx).

 

The results and conclusions of the definitive test are summarized in the following tables: 

Group

Control

Perchlorate d’ammonium

Nominal concentration (mg/L)

 

0

50

88.9

158.1

281.2

500

 

 

 

Geometric mean of measured concentration (mg/L)

0 hour

ND

49.0

86.6

159

298

518

Geometric mean 0-24 h

ND

48.8

82.8

144.9

284.3

503

Geometric mean 0-72 h

ND

37.3

82.1

145.2

285.3

505

 

Inhibition (%)

Growth rate - 24 h

NA

-1.8

0.6

-1.3

4.1

4.8

Growth rate - 48 h

NA

-1.4

5.3

7.6

12.6

17.4

Growth rate - 72 h

NA

0.2

6.9

7.7

16.8

17.6

Yield - 72 h

NA

1.0

32.1

35.0

61.0

62.5

NA: not applicable, ND: not determined

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

72 hours

Growth rate

 

ErC50

> 503

> 503

> 505

ErC10

ND

ND

189.2

(126.6 – 266.8)

Yield

 

EyC50

ND

ND

245.6
(205.1 - 294.8 )

EyC10

ND

ND

42.2

(28.4 - 56.0)

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ECx is the test concentration where effect is reduced by x%.

The 24, 48-hour ErC50 swere not calculated since the inhibition percentage of the average specific growth rate at the highest concentration (500 mg/L) was lower than 50% at the corresponding time (4.8 and 17.4%, respectively).

The 72-hours ErC50 was calculated in the same time that the ErC10 but was not taking in account in the results and the conclusion of this report because this result was not considered as relevant since the inhibition percentage of the average specific growth rateat the highest concentration (500 mg/L) was lower than 50% at this time (17.6%).

The 72-hours ErC10 was calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

The 72-hour EyC50 and EyC10 were calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

Conclusion:

In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 of Perchlorate d’ammonium was > 505 mg/L based on average specific growth rate (ErC50) and 245.6 mg/L based on yield (EyC50).