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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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
(on Day 2 of the test, the light intensity exceeded the mean by more than 15% (16%) and during the first two days of incubation the incubator temperature raised to max. 25.5 °C. This deviation did not adversely impact the results of this study.)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
(on Day 2 of the test, the light intensity exceeded the mean by more than 15% (16%) and during the first two days of incubation the incubator temperature raised to max. 25.5 °C. This deviation did not adversely impact the results of this study.)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
At the start of the test, two samples (50 mL) were taken from the freshly-prepared uninoculated control and test media. After 72 hours, two samples (50 mL) of control and test media were taken from 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)
9.54 mg/10 litres, 15.25 mg/5 litres,9.77 mg/litre, 31.3 mg/1 litre and 100 mg/1 litre was dispersed in dilution medium (algal nutrient medium as recommended in the guidelines) in glass vessels. The contents of each vessel were stirred for approximately 24 hours and then left to stand for approximately 24 hours. Aliquots (800 mL) were then removed mid vessel to provide the Water Accomodated Fractions (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:
no
Total exposure duration:
72 h
Hardness:
25 mg/L as CaCO3
Test temperature:
21.4-25.5 °C
During the first two days of incubation the incubator temperature raised to max. 25.5 °C. At test end, the temperature was 24 °C. As the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.
pH:
test start (0 h): 7.6-7.7
test end (72 h): 8.3-8.7
Nominal and measured concentrations:
nominal loading rates: 0.954, 3.05, 9.77, 31.3 and 100 mg/L as WAF
measured TOC levels:
- loading rates of 0.954, 3.05 and 9.77 mg/L: below the limit of quantification (after background correction)
- loading rates of 31.3 and 100 mg: 0.282 and 2.18 mg C/L at test start and 0.0837 and 1.40 mg C/L at test end;
overall mean measured TOC levels: 0.154 and 1.75 mg C/L
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: 1x10⁴ cells/mL
- Control end cells density: 118 x10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- 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: 1.0 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 6148 to 6710 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.
On Day 2, the light intensity exceeded the mean by 16%. As the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.

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
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth after 72 hours was 21% at 10 mg/L and 29% at 100 mg/L
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:
growth rate
Remarks on result:
other: results are expressed in terms of loading rates
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: yield
Remarks on result:
other: results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: results are expressed in terms of loading rates
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
45.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: 11-95 mg/L; results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
55.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits: 20-95 mg/L; results are expressed in terms of loading rates
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
31.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, yield and biomass
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 was observed (see Table 2). This had no influemce on calculation of EC50 values.

At the start of the test, the test media (as WAF) at all nominal loading rates were colourless dispersions.
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:
Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates. 95% confidence intervals were calculated using the likelihood ratio method. Williams’ test was used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test was used.

Table 1: Cell densities

 

Nominal loading rates

(mg/L)

Replicate

number

Cell densities (cells/mL)

24 hours

48 hours

72 hours

 

 

 

 

 

Control

R1

53450

243283

1111300

 

R2

50717

251717

1109933

 

R3

48217

249250

1196100

 

R4

48350

242450

1115600

 

R5

49983

259017

1301333

 

R6

58117

297350

1253033

 

Mean

51472

257178

1181217

 

 

 

 

 

0.954

R1

49517

262483

991400

 

R2

48783

266350

1007267

 

R3

54917

255350

1415433

 

Mean

51072

261394

1138033

 

 

 

 

 

3.05

R1

54950

265950

1027733

 

R2

58817

291683

1512400

 

R3

64517

297650

1438600

 

Mean

59428

285094

1326244

 

 

 

 

 

9.77

R1

54950

259950

1257233

 

R2

54183

273483

1225033

 

R3

52983

262450

1254367

 

Mean

54039

265294

1245544

 

 

 

 

 

31.3

R1

55683

282050

1252333

 

R2

50783

247717

929967

 

R3

49750

301417

1224833

 

Mean

52072

277061

1135711

 

 

 

 

 

100

R1

42383

194783

848167

 

R2

42283

178650

731600

 

R3

46150

204550

870933

 

Mean

43606

192661

816900

 

 

 

 

 

 

R1-R6: replicate number

Note: the initial cell density was estimated to be 0.865 x 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

21.0

0.0

-

 

0.0954

3

20.6

2.0

>0.999W

 

0.305

3

23.6

-12.4

>0.999W

 

0.977

3

22.0

-4.9

>0.999W

 

3.13

3

20.9

0.3

>0.999W

 

10

3

14.9

29.1

0.001**W

 

 

 

 

 

 

 

 

Growth rate to

72 hours

Control

6

0.066

0.0

-

 

0.0954

3

0.066

1.0

>0.999W

 

0.305

3

0.068

-2.2

>0.999W

 

0.977

3

0.067

-1.2

>0.999W

 

3.13

3

0.066

1.0

0.832W

 

10

3

0.061

7.8

0.001**W

 

 

 

 

 

 

 

Growth rate

0 to 24 hours

Control

6

 0.068

 0.0

-

0.0954

3

 0.068

 0.4

>0.999D

0.305

3

 0.074

 -8.8

0.025*D

0.977

3

 0.070

 -3.1

0.731D

3.13

3

 0.069

 -0.8

>0.999D

10

3

 0.061

 10.1

0.010**D

 

 

 

 

 

 

Growth rate

24 to 48 hours

Control

6

0.067

0.0

-

0.0954

3

0.068

-1.6

>0.999W

0.305

3

0.065

2.4

>0.999W

0.977

3

0.066

1.1

>0.999W

3.13

3

0.070

-3.8

>0.999W

10

3

0.062

7.7

0.018*W

 

 

 

 

 

 

Growth rate

48 to 72 hours

Control

6

0.064

0.0

-

0.0954

3

0.061

4.4

>0.999W

0.305

3

0.064

0.0

>0.999W

0.977

3

0.064

-1.4

>0.999W

3.13

3

0.059

7.9

0.289W

10

3

0.060

5.4

0.289W

 

 

 

 

 

 

Yield

Control

6

1171217

0.0

-

 

0.0954

3

1128033

3.7

>0.999W

 

0.305

3

1316244

-12.4

>0.999W

 

0.977

3

1235544

-5.5

>0.999W

 

3.13

3

1125711

3.9

0.899W

 

10

3

806900

31.1

0.005**W

 

p values are for the comparison with Control using Williams' test (W) and Dunnett's test (D)

*p< 0.05, **p< 0.01

 

 

Control cultures:

-cell concentration increased by a factor of 118;

-the mean coefficient of variation for daily growth rates ranged between 3.0 and 4.4%;

- the coefficient of variation for the average specific growth rates was 1.5% 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: 31.3 mg/L, EC10: >100 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
100 mg/L
EC10, LC10 or NOEC for freshwater algae:
31.3 mg/L

Additional information

The EC50/LC50 for the test material is much higher than its water solubility.