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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
Study period:
2010-07-09 to 2010-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restrictions. The study was conducted according to the OECD Guideline 201, and in compliance with GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Loading rates: 0 (Control), 5.0, 25 and 100 mg/L

- Sampling method: Samples of the control and WAFs were taken for TOC analysis (TOC = Total Organic Carbon) and GC-MS analysis (GC-MS = Gas Chromatography - Mass Spectrometry) from separate vessels at 0 h and 72 h to assess the stability of exposure concentrations.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The test item Crude Sulfate Turpentine (S: 0.02 %) is a complex mixture with a low water solubility. To avoid adverse physical effects of undissolved or emulsified test material on the algae, the Alga Test with Crude Sulfate Turpentine (S: 0.02 %) was carried out with aqueous extracts (WAF = Water Accommo-dated Fraction) at various loading rates of the test item in culture medium.

In a preliminary study with a test item of comparably low water solubility (Sylva-blend TM PF 40, STZ No. 01-05-001; study sponsor Arizona Chemical), the durations of the stirring and phase separation phases of WAF preparation were investigated. Water Accommodated Fractions were prepared and analysed following a range of mixing and settling periods to ensure optimal equilibrium and phase separation of the test item in the WAFs. Quantitation of dissolved component concentrations was achieved by TOC analysis (TOC = Total Organic Carbon).

Taking into account the results of this preliminary WAF-study, the WAFs for the Alga Test Test with Crude Sulfate Turpentine (S: 0.02 %) were prepared by stirring various amounts of the test item in culture medium for 24 h with magnetic stirrers. The WAFs were prepared in closed glass vessels. The test load-ing rates of Crude Sulfate Turpentine (S: 0.02 %) were weighed on weighing scoops, that afterwards were placed into the glass vessels. The vessels were then filled with culture medium and closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. To ensure this, the vortex developed at the surface by stirring was set at approximately 10 % of the water depth. After stirring for 24 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation. The extracts gained with this method were clear.

The Control (culture medium) for the Alga Test was treated in the same way as the WAFs.

The vessels used for Exposure Concentration Analysis, including the Control and WAFs, were kept under the same conditions as the test vessels.

- Controls: algal growth medium
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM

- Common name: Green algae.

- Strain: SAG 86.81

- Source: Albrecht von Haller Institut für Pflanzenwissenschaften der Universität Göttingen.

- Age of inoculum: 72 h.

- Method of cultivation: on agar tubes (OECD Medium, 0.8% Agar), transferred to a new agar tube every 2 months.


ACCLIMATION

- Acclimation period: 72 h.

- Culturing media and conditions: same as test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not applicable
Test temperature:
25 ± 2°C
pH:
7.8 - 7.9 at time 0 h, in the range of 8.0–10.4 at time 72 h.

The increase of pH is caused by the algal growth in the test vessels. In common with other species of freshwater algae, Desmodesmus subspicatus gains CO2 from HCO3-, leading to a release of hydroxide ions that raise the alkalinity of the medium.

The shift in pH of >1 unit is indicative of good algal growth and is not considered to invalidate the test result.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal loading rates: 0 (Control), 5, 10, 25, 50 and 100 mg/L as WAFs.

TOC-values of the Alga Test (Table 3) confirmed the low water solubility of the test item. The deviations of the TOC-values in the control and at test loading rates of 5.0 mg/L and 25 mg/L lie within or very close to the confidence-limits (± 0.3 mg C/L) of these measurements. The TOC-value at test loading rate of 100 mg/L decreased to 80 % of the initial value. Therefore the WAFs were considered to remain approximately stable over the 72-h test period.

GC-MS analysis of the Control medium and the WAFs in the Alga Test (Table 4) revealed that the total concentrations of fingerprint components decreased to 19 % of initial values at test loading rate 5.0 mg/L, to 24 % of initial values at test loading rate 25 mg/L and to 25 % of initial values at test loading rate 100 mg/L over 72 h. This finding is probably caused by biodegradation of these components during 72 h.

Quantitative GC-MS-values were definitely lower compared to TOC-values. This is caused by the fact that only fingerprints of the sample were detected by GC-MS analysis whilst TOC analysis detected Total Organic Carbon as a sum parameter.

The results of the test were reported and interpreted with reference to nominal loading rates.
Details on test conditions:
TEST SYSTEM

- Test vessel: Erlenmeyer flasks with ground-in glass stoppers

- Material, size, headspace, fill volume: glass 250 ml with 200 mL of test medium.

- Aeration: None

- Renewal rate of test solution: none

- Initial cells density: ca. 2500 cells/mL

- Control end cells density: average over 3 vessels was 570,000 cells/mL

- No. of vessels per concentration: 3

- No. of vessels per control: 3


GROWTH MEDIUM

- Standard medium used: yes

The following stock solutions were prepared in deionised water according to OECD-Guideline 201 (final concentrations in the test solutions in brackets):

Solution 1:
NH4Cl: 1.5 g/L (15 mg/L)
MgCl2 x 6 H2O: 1.2 g/L (12 mg/L)
CaCl2 x 2 H2O: 1.8 g/L (18 mg/L)
MgSO4 x 7 H2O: 1.5 g/L (15 mg/L)
KH2PO4: 0.16 g/L (1.6 mg/L)

Solution 2:
FeCl3 x 6 H2O: 0.08 g/L (0.08 mg/L)
Na2 EDTA x 2 H2O: 0.1 g/L (0.01 mg/L)

Solution 3:
H3BO3: 0.185 g/L (0.185 mg/L)
MnCl2 x 4 H2O: 0.415 g/L (0.415 mg/L)
Na2MoO4 x 2 H2O: 0.0007 g/L (7 x 10-3 mg/L)
ZnCl2: 3.0 mg/L (3 x 10-3 mg/L)
CoCl2 x 6 H2O: 1.5 mg/L (1,5 x 10-3 mg/L)
CuCl2 x 2 H2O: 0.01 mg/L (10-5 mg/L)

Solution 4:
NaHCO3: 50.0 g/L (50 mg/L)

The culture medium was prepared by adding 10 mL of stock solution 1 and 1 mL of stock solution 3 per litre of deionised water. The mixture was then autoclaved at 121 °C for 15 minutes. Solution 2 + 4 (1 mL/L) were added after autoclaving by sterile filtration (0.45 µm pore size). The pH-value of the culture medium was 7.7 - 8.0.


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: culture medium and deionised water.

- Total organic carbon: See Table 3.

- Culture medium different from test medium: no

- Intervals of water quality measurement: beginning and end of experiment (0h and 72 h).


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 8000 to 8200 Lux using cool-white light and maximum ± 15 % variation within the testing area, permanent illumination


EFFECT PARAMETERS MEASURED:

- Determination of cell concentrations: microscopy with counting chambers at time 24 h, 48 h and 72 h.


TEST CONCENTRATIONS

- Range finding study: None
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
17.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fractions (WAFs)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
16.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fractions (WAFs)
Basis for effect:
biomass
Remarks:
(yield)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fractions (WAFs)
Basis for effect:
other: growth rate and biomass (yield)
Details on results:
- Exponential growth in the control: yes, the cell concentration in the control cultures increased by a factor of 230 in 72 h.

- Effect concentrations exceeding solubility of substance in test medium: the loading rates at which the water-accommodated fractions were prepared exceeded the solubility of the test substance.
Reported statistics and error estimates:
The results of the Alga Test were analysed statistically with reference to the specific growth rate [µ] and biomass (yield).

Normality of the data was tested using a Kolmogorov-Smirnov-test (α = 0.05).

Homogeneity of variances was tested by Bartlett`s test (α = 0.05).

For all measured end values, statistical differences among treatment groups were determined using a one-way analysis of variance (ANOVA).

Two-sided Dunnet's procedure for multiple comparison was used to determine, which concentration of the test item differed significantly from the control (p = 0.05).

The results of the two-sided Dunnet's test (p = 0.05) were used to determine the NOELR (No Observable Effect Loading Rate) of the specific growth rate [µ] and of the biomass (yield).

The NOELR (No Observable Effect Loading Rate) is defined as the highest test loading rate, where inhibition of the specific growth rate [µ] and of the biomass was statistically not significantly different when compared to the control.

Table 1. Test results – average specific growth rates (µ) over the test period 0-72 hours

Loading rate

[mg/L]

Cells / mL

Average

Specific

growth rate

[µ]

Average

Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation

Vc [%]

Inhibition of Average

Specific

growth rate

[%]

Inhibition of Average

Specific

growth rate

Mean value

[%]

Control 1

576000

1.81

1.81

0.00

0.15

 

 

Control 2

568000

1.81

Control 3

576000

1.81

5.0

584000

1.82

1.81

0.01

0.45

-0.34

-0.08

5.0

560000

1.80

0.43

5.0

584000

1.82

-0.34

10

568000

1.81

1.80

0.01

0.68

0.17

0.79

10

528000

1.78

1.52

10

552000

1.80

0.70

25

5000

0.23

0.15

0.09

57.1

87.3

91.8

25

3000

0.06

96.7

25

4000

0.16

91.4

50

4500

0.20

0.10

0.10

95.7

89.2

94.3

50

2500

0.00

100

50

3500

0.11

93.8

100

3000

0.06

0.09

0.06

59.7

96.7

94.9

100

3000

0.06

96.7

100

4000

0.16

91.4

 Remark: A negative inhibition value means an enhancement of the parameter.

Table 2. Test results – biomass (yield) over the whole test period 0-72 hours

Loading rate

[mg/L]

Cells/mL

Yield

Cells/mL

Yield

Cells/mL

Mean value

Standard deviation

Coefficient of variation

Vc [%]

Inhibition

of Yield [%]

Inhibition of Yield [%] Average

Control 1

576000

573500

570833

4619

0.81

 

 

Control 2

568000

565500

Control 3

576000

573500

5.0

584000

581500

573500

13856

2.42

-1.87

-0.47

5.0

560000

557500

2.34

5.0

584000

581500

-1.87

10

568000

565500

546833

20133

3.68

0.93

4.20

10

528000

525500

7.94

10

552000

549500

3.74

25

5000

2500

1500

1000

66.7

99.6

99.7

25

3000

500

99.9

25

4000

1500

99.7

50

4500

2000

1000

1000

100

99.7

99.8

50

2500

0

100

50

3500

1000

99.8

100

3000

500

833

577

69.3

99.9

99.9

100

3000

500

99.9

100

4000

1500

99.7

 Remark: A negative inhibition value means an enhancement of the parameter.

Table 3. Results of TOC analysis of the test media

Time [h]

Control

5.0 mg/L

25 mg/L

100 mg/L

0

0.3

0.3

1.7

4.7

72

0.3

0.1

1.2

3.8

 

Table 4. Results of GC-MS analysis of test media

Vessel

Time [h]

a-Terpineol
[µg/L]

a-Pinene
[µg/L]

b-Pinene
[µg/L]

3-Carene
[µg/L]

Limonene
[µg/L]

Total

[µg/L]

Control

0

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

Control

72

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

5.0 mg/L

0

5.81

94.1

10.1

23.2

9.12

142

5.0 mg/L

72

3.76

11.1

2.32

7.12

2.82

27.1

25 mg/L

0

37.6

735

75.3

222

96.1

1166

25 mg/L

72

22.5

116

22.8

84.5

38.4

285

100 mg/L

0

125

1111

116

243

113

1708

100 mg/L

72

104

173

32.7

81.6

41.5

432

Remarks:

*   Limit of detection

**   derived from equation of the calibration line (axis intercept); no signal was measured in the controls for α-Terpineol, β-Pinene and Limonene

         Quantitative GC-MS-values are based on results and/or limits of detection
Validity criteria fulfilled:
yes
Conclusions:
A 72h EL50 of 17.1 mg/L and NOELR of 10 mg/L have been determined for the effect of the test substance on growth rate of Desmodesmus subspicatus.

Description of key information

A 72h-EL50 of 17.1 mg/l and NOELR of 10 mg/l have been determined for the effect of the test substance on growth rate of Desmodesmus subspicatus. The substance had a sulfur content of 0.02%. A 72h-EL50 of 22.5 mg/l and NOELR of 5 mg/l have been determined for the same endpoint in a second test conducted on a sample of the substance with a sulfur content of 3.6%. It is therefore concluded that the two samples were equally toxic to aquatic algae.

Short-term toxicity data are also available for alpha- and beta-pinene, which are both constituents of block 1 of TOPP. The 48h-LOEC for alpha-pinene was determined as 0.494 mg/l (and the NOEC was estimated to be around 0.25 mg/l) and the 48h-EC50 and 48h-EC10 for beta-pinene was determined to be 0.83 mg/l and 0.38 mg/l respectively. However, significant loss of test substance was observed in both tests (presumably a result of biodegradation in and/or volatilisation from the static test system) and so these values may not represent the true toxicity of alpha- and beta-pinene.

Key value for chemical safety assessment

EC50 for freshwater algae:
17.1 mg/L
EC10 or NOEC for freshwater algae:
5 mg/L

Additional information