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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the start of the test, four samples (50 mL) were taken from freshly-prepared uninoculated control and test media.
After 72 hours, four samples (50 mL) of control and test media were taken from the pooled contents of uninoculated replicate flasks.
Samples were stored in a freezer prior to analysis.
Vehicle:
no
Details on test solutions:
REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
200 mg test material was dispersed in 2 L dilution medium (algal nutrient medium as recommended in the guidelines) in a glass vessel. The contents of the vessel were stirred for approximately 24 hours in the dark and then left to stand overnight. An aliquot (1200 mL) was then removed mid vessel to provide the Water Accomodated Fraction (WAF).
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), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth.

ACCLIMATION
- Acclimation period: 6 days (pre-culturing)
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
25 mg/L as CaCO3
Test temperature:
21 - 23 °C
pH:
test start (0 h): 7.7
test end (72 h): 8.3
Nominal and measured concentrations:
nominal loading rates: 100 mg/L as WAF
measured levels at test start: 0.11 - 0.45 mg C/L (mean: 0.28 mg C/L)
measured levels at test end: the mean measured level of TOC in the test medium was below that found in the control sample; so it was not possible to quantify the level of carbon that was attributable to the test substance
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: 10^4 cells/mL
- Control end cells density: 115 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 7.4 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 6572 to 6716 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no inhibition after 72 h
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, biomass and yield
Remarks on result:
other: results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, biomass and yield
Remarks on result:
other: results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, biomass and yield
Remarks on result:
other: results are expressed in terms of loading rates
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: minor stimulation of daily growth rates was observed (4%) at Day 3 yo 4 (Tables 1 and 2).

At the start of the test, the test medium was a colourless dispersion.

Inhibition of growth rate, biomass (area under curve, AUC) and yield ranged between -4 and 18% (Tables 1 and 2). Although statistically significant, the inhibition observed was not considered to be of biological significance since it was <20%.
Results with reference substance (positive control):
- 72-hour EbC50: 0.541 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)
Reported statistics and error estimates:
The t-test was used to compare the treated group with the control group.
Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002).

Table 1: Cell densities

 

Nominal loading rates

(mg/L)

Replicate

number

Cell densities (cells/mL)

24 hours

48 hours

72 hours

 

 

 

 

 

Control

R1

43100

238533

1131517

 

R2

45000

273300

1178517

 

R3

43700

240933

1115583

 

R4

46367

261000

1142117

 

R5

44200

252933

1123917

 

R6

46667

248600

1225083

 

Mean

44839

252550

1152789

 

 

 

 

 

100

R1

34533

195433

959783

 

R2

36600

200733

990883

 

R3

37400

193800

992617

 

R4

32400

199267

959883

 

R5

37567

208500

995483

 

R6

35300

207633

956617

 

Mean

35633

200894

975878

 

 

 

 

 

 

R1-R6: replicate number

Note: the initial cell density was estimated to be 0.695x 104/mL

 

Table 2  Inhibition of growth

Parameter

Nominal loading

rates

(mg/L)

Sample size

Mean

Inhibition

(%)

p-value

 

Area under curve to 72 hours

Control

6

20.4

0.0

-

 

100

6

16.8

17.6+

<0.001***

 

 

 

 

 

 

 

 

Growth rate to

72 hours

Control

6

0.066

0.0

-

 

100

6

0.064

3.5+

<0.001***

 

 

 

 

 

 

 

Growth rate

0 to 24 hours

Control

6

0.063

0.0

-

100

6

0.053

15.4+

<0.001***

 

 

 

 

 

 

Growth rate

24 to 48 hours

Control

6

0.072

0.0

-

100

6

0.072

-0.1

0.934

 

 

 

 

 

 

Growth rate

48 to 72 hours

Control

6

0.063

0.0

-

100

6

0.066

-4.1+

0.036*

 

 

 

 

 

 

Yield

Control

6

1142789

0.0

-

10

6

965877.7

15.5+

<0.001***

 

pvalues are for the comparison with Control using thet-test

*p< 0.05, ***p< 0.001

+inhibition was not considered to be a biologically significant effect (<20%)

 

Control cultures:

-cell concentration increased by a factor of 115;

-the mean coefficient of variation for daily growth rates ranged between 2.1 and 3.4%;

-the coefficient of variation for the average specific growth rates was 0.75% during the 72 hour exposure period.

 

Validity criteria fulfilled:
yes
Remarks:
The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD TG 201, EU C.3):
EC50: >100 mg/L, NOELR: ≥ 100 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The EC10/LC10 for the test material is much higher than its water solubility.