Registration Dossier

Diss Factsheets

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:
17 June 2010 to 27 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 of the study and the results); adequate coherence between data, comments and conclusions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the temperature varied by more than ± 2°C during the test, maximum temperature during the test was higher than 24°C, the pH varied by more than 1.5 units in the control during the test
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
as mentionned above
Qualifier:
according to guideline
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of barium chromate, copper dichromium tetraoxide and copper oxide
EC Number:
701-287-1
Molecular formula:
CuO, CuCr2O4, BaCrO4
IUPAC Name:
Reaction mass of barium chromate, copper dichromium tetraoxide and copper oxide
Details on test material:
- Name of test material (as cited in study report): Chromite de cuivre
- Substance type: multiconstituents
- Physical state: black powder
- Composition of test material, percentage of components: 34% of copper, 8.5% of baryum, 31% of chrome
- Purity test date: 11 January 2010
- Lot/batch No.: 09/0177
- Expiration date of the lot/batch: 17 August 2014
- Storage condition of test material: in darkness at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Combined range-finding and limit test:
At T0 hour, duplicate samples of 5 mL were taken from the limit test solution (limit of solubility/saturation (LS)) just before distribution in the test vessel.
At T24, T48 and T72 hours, samples were taken from each limit test solution replicate, and then pooled in order to obtain two samples of 5 mL.
Further samples were taken at T24, T48 and T72 hours from the limit test solution 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.
Although samples were taken during the test, no chemical analysis was performed on these samples because the test item was found to be toxic.

Definitive test:
Two samples of 10 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 6 mL for T24 and T48 hours and two samples of 10 mL at T72 hours .
Two samples of 10 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.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the test solutions:
The stock solutions for the combined range-finding and limit test and definitive tests, were prepared by dispersing the test item directly in OECD medium.
The conditions of preparation of the stock solutions are reported in the following table:

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

Combined range
finding and limit test 100 1000 100 23h30 min

Definitive test 100 1000 100 23h30min

As the test item is not soluble in water, the stock solutions were prepared by stirring an excess amount of the test item (100 mg/L) in test medium for up to 24 hours. As test item particles were still in suspension after agitation the stock solution was filtered through a filter of porosity 0.22 µm. to remove the suspended fraction. The filtered solution, corresponding to the limit of solubility/saturation of the test item under our experimental conditions (LS), was then used as a stock solution to prepare the test solutions under agitation.Filtration on filter 0.22 µm was chosen because the certificate of analysis indicates that 50% of the particles constituting the test item are < 0.43 µm.

Test solutions were prepared by further dilution of the stock solution (LS) with OECD medium to provide a geometric series of concentrations:
- LS/10000, LS/1000, LS/100, LS/10 and LS for the combined range finding and limit test,
- LS/16, LS/8, LS/4, LS/2 and LS 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 the pH was therefore not adjusted 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 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: at CIT. The algae are cultured under sterile conditions and maintained at exponential growth rate.

ACCLIMATION
- Acclimation period: none

Study design

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

Test conditions

Hardness:
32.1 mg/L as CaCO3
Test temperature:
between 23.7°C and 27.6°C
pH:
between 8.07 and 9.89
Dissolved oxygen:
Not measured
Salinity:
Not measured
Nominal and measured concentrations:
Measured concentrations in the solutions with and without algae were within +/- 20% of the initial value (T0 hours) throughout the test. The test item was therefore stable under our experimental conditions and the study results could be based on initial concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass test vessels (250 mL Erlenmeyer flasks)
- Type (delete if not applicable): closed
- 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.
- Initial cells density: 5 x 103 cells/mL
- Control end cells density: between 160.00 x 104 cell/mL and 256.00x 104 cell/mL
- No. of vessels per concentration (replicates): limit test: 6 replicates (with algae) and 1 replicate (without algae); range-finding test: 2 replicates (with algae), definitive test: 3 replicates (with algae) and 1 replicate (without algae)
- No. of vessels per control (replicates): combined range-finding and limit test and definitive test: 6 replicates (with algae)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes. 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. Test medium: OECD medium adapted for the test item that contains metal substance. Reconstituted water OECD medium prepared using sterile water with a conductivity of 10 µS/cm.
- Intervals of water quality measurement: the pH was measured in the control and in the limit test concentration and all test concentrations of the definitive test. It was measured at the beginning of the test, in the preparation test container, before distribution in test vessels and at the end of the test in one replicate. The temperature was measured between 0 and 2 hours after the beginning of the test in a flask containing OECD medium but no algae run alongside the test. The maximun and minimum temperatures were also recorded in this flask 24, 48 and 72 hours after the start of the test.
Hardness was measured once in OECD medium before the start of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: the pH of all test solutions remained within the range 6.0 +/- 0.2 to 9.0 +/- 0.2 after preparation and the pH was therefore not adjusted before addition of the algae
- Photoperiod: 24 hours light per day
- Light intensity and quality: between 7020 lux and 7590 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Malassez cell Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: for the combined range-finding and limit test the ratio between two consecutive concentrations was a constant factor not exceeding 10; for the definitive test, a geometric series of concentrations with a ratio between two consecutive concentrations ≤ 2.2 and not exceeding 3.2 was used.

- Combined range finding and limit test
- Test concentrations: LS, LS/10, LS/100, LS/1000, LS/10000
- Results used to determine the conditions for the definitive study: average specific growth rate inhibitions at LS/10000, LS/1000, LS100 and LS/10 were 0.12, 2.01, 31.14 and 4.15%, respectively, relative to the control, after 72 hours. Yield inhibitions at LS/10000, LS/1000, LS100 and LS/10 were 1.69, 12.3, 18.1 and 22.9%, respectively, relative to the control, after 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Results with reference substance (positive control):
The result and conclusion of the last reference test (relative to the experimental completion date of this study) are presented below:
- Study No.: 36865 EAA (GLP study)
- Dates of the test: April 2010
- Potassium dichromate: . supplier: Acros Organics
. batch No.: A0243103
- 72-hour ErC50: 1.22 mg/L of potassium dichromate
- Conclusion: ErC50 accepted.

Any other information on results incl. tables

The 24, 48 and 72-hour ErCx and the72-hour EyCx were estimated from the % inhibition of average specific growth rate (ErCx and yield (EyCx).

They were calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

The results of the definitive test based on the nominal concentrations are summarized in the following tables:

 

Group

Control

Chromite de cuivre

 

Nominal concentration

Concentration

0 mg/L

LS/16

LS/8

LS/4

LS/2

LS

 

 

 

 

 

measured concentration (mg/L)

Concentration

ND

0.16

0.32

0.62

1.26

2.63

 

 

 

Inhibition (%)

Growth rate - 24 h

NA

2.14

13.6

50.1

75.4

96.8

Growth rate - 48 h

NA

-0.09

15.0

57.1

88.8

100

Growth rate - 72 h

NA

1.45

8.53

47.2

73.7

95.4

Yield - 72 h

NA

8.90

38.7

93.9

98.9

99.9

ND  : not determined, NA: not applicable

 

The conclusions of the definitive test are summarized in the following table:

 

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

72 hours

Growth rate

 

ErC50

0.69

(0.62 - 0.77)

0.59

(0.54 - 0.64)

0.75

(0.68 -0.84)

ErC10

ND

ND

0.30

(0.25 – 0.35)

Yield

 

EyC50

ND

ND

0.34

(0.31 – 0.37)

EyC10

ND

ND

0.18

(0.15 – 0.20)

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ExC10 is the test concentration where effect is reduced by 10%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 of Chromite de cuivre was 0.75 mg/L based on average specific growth rate (ErC50) and 0.34 mg/L based on yield (EyC50).
Executive summary:

Methods

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

Combined range-finding and limit test

.           Limit test: two groups of six replicate algal suspensions were exposedto the nominalconcentrations of 0 (control) and limit of solubility/saturation (LS) for 72 hours. Cell growth data were recorded at 24, 48 and 72 hours,

.           Range-finding test: four groups of two replicate algal suspensions were exposed to nominal concentrations of 0.01, 0.1, 1 and 10 mg/L of the test item under the same experimental conditions.

 

Definitive test

In the definitive test, algal suspensions were exposed to the nominal concentrations of LS/16, LS/8, LS/4, LS/2 and LS mg/L for 72 hours.

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

 

Chemical analysis

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

Validity criteria

All study validity criteria were respected for each test:

.           the cell concentration in the control cultures increased by a factor of at least 16 within 72 hours,

.           the mean coefficient of variation of daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) did not exceed 35%,

.           the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

 

Chemical analysis

Measured concentrations in the solutions with and without algae were within ± 20% of the initial value (T0 hours) throughout the test.The test item was therefore stable under our experimental conditions and the study results could be based on initial concentrations.

Inhibition of cell growth

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

They were calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

 

The results of the definitive test based on the nominal concentrations are summarized in the following tables:

Group

Control

Chromite de cuivre

 

Nominal concentration

Concentration

0 mg/L

LS/16

LS/8

LS/4

LS/2

LS

 

 

 

 

 

measured concentration (mg/L)

Concentration

ND

0.16

0.32

0.62

1.26

2.63

 

 

 

Inhibition (%)

Growth rate - 24 h

NA

2.14

13.6

50.1

75.4

96.8

Growth rate - 48 h

NA

-0.09

15.0

57.1

88.8

100

Growth rate - 72 h

NA

1.45

8.53

47.2

73.7

95.4

Yield - 72 h

NA

8.90

38.7

93.9

98.9

99.9

ND  : not determined, NA: not applicable

 

The conclusions of the definitive test are summarized in the following table:

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

72 hours

Growth rate

 

ErC50

0.69

(0.62 - 0.77)

0.59

(0.54 - 0.64)

0.75

(0.68 -0.84)

ErC10

ND

ND

0.30

(0.25 – 0.35)

Yield

 

EyC50

ND

ND

0.34

(0.31 – 0.37)

EyC10

ND

ND

0.18

(0.15 – 0.20)

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits), ExC10 is the test concentration where effect is reduced by 10%.

 

Conclusion

In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 of Chromite de cuivre was 0.75 mg/L based on average specific growth rate (ErC50) and 0.34 mg/L based on yield(EyC50).

Categories Display