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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:
Samples from control and test media were taken from freshly prepared uninoculated media at test start. At test end (72 hours) samples were taken from uninoculated replicate flasks incubated under the test conditions.
Samples were analysed without prior storage.
Vehicle:
no
Details on test solutions:
REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
Aliquots of the test substance were dispersed in dilution medium. The bulk preparations were stirred for approximately 24 hours in the dark, then left to stand for approximately 24 hours in the dark. An aliquot (3 L) was then syphoned from a mid-vessel location of each preparation vessel, of which the first litre was discarded, to give 2 L aliquots of Water Accommodated Fractions (WAF) which were used as test media with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg/L. The lowest nominal loading rate of 0.32 mg/L was prepared by dilution of an aliquot of the WAF at 1.0 mg/L.
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:
21.7-22.8 °C
pH:
test start (0 h): 7.79-7.92
test end (72 h): 7.91-8.82
Nominal and measured concentrations:
nominal loading rates: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L as WAF
measured levels: The measured levels of TOC were below the limit of quantification of the analysis method after background correction for the control medium.
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: 150x10⁴ 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: 0.98 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 5648 to 5736 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. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts. Nominal loading rates 0, 0.32, 1.0 and 3.2 mg/L were corrected against un-inoculated dilution media. The nominal loading rates of 10, 32 and 100 mg/L were corrected against un-inoculated WAF at the same loading rate as the test flask, as the initial cell counts for these treatment groups were higher than the background cell counts, indicating that the WAF’s at these levels were effecting the cell counts.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth after 72 hours was 82% and 71% at 100 and 10 mg/L; no inhibition occurred at 1.0 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
46.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 28-89 mg/L; results are expressed in terms of loading rates
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 0.2-2.5 mg/L; results are expressed in terms of loading rates
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 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:
EL50
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: 4.1-5.9 mg/L; results are expressed in terms of loading rates
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: 1.1-2.8 mg/L; results are expressed in terms of loading rates
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.32 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
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: no biologically significant stimulation of growth was observed (see Tables 1 and 2).

At the start of the test, the test media were colourless.
Results with reference substance (positive control):
- 72-hour ErC50: 0.73 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)
Reported statistics and error estimates:
For determination of the ECx values the approaches recommended in the OECD guideline 201 were used. 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

45583

237267

1392783

 

R2

48150

263800

1538617

 

R3

46783

242233

1444350

 

R4

45517

239067

1512217

 

R5

49617

295900

1592783

 

R6

51117

266567

1549983

 

Mean

47794

257472

1505122

 

 

 

 

 

0.320

R1

53317

288133

1637750

 

R2

51350

280433

1578150

 

R3

51483

282900

1525150

 

Mean

52050

283822

1580350

 

 

 

 

 

1.00

R1

50517

280167

1333550

 

R2

52083

259567

1293583

 

R3

45017

225333

1188017

 

Mean

49206

255022

1271717

 

 

 

 

 

3.20

R1

35417

186333

1036083

 

R2

43783

198933

1119417

 

R3

51950

245767

1247917

 

Mean

43717

210344

1134472

 

 

 

 

 

10.0 A

R1

7700

40267

199100

 

R2

7133

32100

152767

 

R3

10267

48100

196100

 

Mean

8367

40156

182656

 

 

 

 

 

32.0 A

R1

5133

17567

99200

 

R2

7567

32667

126867

 

R3

8733

39600

159733

 

Mean

7144

29944

128600

 

 

 

 

 

100 A

R1

-3000 B

19833

145600

 

R2

-8633 B

14133

137433

 

R3

-8967 B

-633

103333

 

Mean

-6867 B

11111

128789

 

 

 

 

 

R1-R6   Replicate number.

Note: Initial nominal cell density = 0.974 x 104cells/mL (Equipment used: Coulter Z Series Particle Count and Size Analyser)

A: Nominal loading rates of 10, 32 and 100 mg/L were corrected against un-inoculated WAF at the same loading rate as the test flask

B: Results were below the background level for the un-inoculated WAF at this concentration, therefore the values reported were negative, and statistical analysis could not be performed for this data.

 

Table 2  Inhibition of growth

 

Parameter

Nominal loading

rates

(mg/L)

Sample size

Mean

Inhibition

(%)

p-value

 

 

 

 

 

 

Growth rate to

72 hours

Control

6

0.0696

0.0

 

0.32

3

0.0703

-1.0

1.000W

1.0

3

0.0673

3.4

0.082W

3.2

3

0.0657

5.7

0.005**W

10

3

0.0402

42.2

<0.001***W

32

3

0.0352

49.4

<0.001***W

100

3

0.0353

49.2

<0.001***W

 

 

 

 

 

 

Growth rate

0 to 24 hours

Control

6

0.0651

0.0

 

0.32

3

0.0687

-5.5

1.000W

1.0

3

0.0663

-1.8

1.000W

3.2

3

0.0610

6.4

0.444W

10

3

-0.0080

112.2

<0.001***W

32

3

-0.0150

123.1

<0.001***W

100 A

3

A

A

A

 

 

 

 

 

 

Growth rate

24 to 48 hours

Control

6

0.0701

0.0

 

0.32

3

0.0707

-0.9

1.000W

1.0

3

0.0685

2.3

0.592W

3.2

3

0.0657

6.3

0.088W

10

3

0.0653

6.8

0.066W

32

3

0.0584

16.7

<0.001***W

100

3

-0.1105

257.6

<0.001***W

 

 

 

 

 

 

Growth rate

48 to 72 hours

Control

6

0.0737

0.0

 

0.32

3

0.0715

2.9

0.969D

1.0

3

0.0671

9.0

0.208D

3.2

3

0.0704

4.4

0.824D

10

3

0.0634

13.9

0.021*D

32

3

0.0623

15.5

0.010**D

 

100

3

0.0889

-20.7

0.003**D

 

 

 

 

 

 

Yield to

72 hours

Control

6

1495122

0.0

 

0.32

3

1570350

-5.0

1.000W

1.0

3

1261717

15.6

<0.001***W

3.2

3

1124472

24.8

<0.001***W

10

3

172656

88.5

<0.001***W

32

3

118600

92.1

<0.001***W

100

3

118789

92.1

<0.001***W

 

 

 

 

 

 

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

* p<0.05, ** p<0.01, *** p<0.001

Note: Negative values indicate an increase in algal growth above that of Control growth

A: Results were negative; therefore statistical analysis was not performed (see Table 1, footnote B).

 

Control cultures:

- cell concentration increased by a factor of 150;

- the mean coefficient of variation (CoV) for section by section daily growth rates was 6.7%;

- the coefficient of variation for the average specific growth rates was 0.987% 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 201, EU C.3):

EL50: 46.5 mg/L, NOELR: 1.0 mg/L, EL10: 0.85 mg/L

Key value for chemical safety assessment

Additional information

The results indicate that the test material is not inhibitory to growth of algae up to the limits of its water solubility (<1 mg/L) and a NOEC and thus a PNEC cannot be determined.