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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:
From 04 to 08 February 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 201. However, pH level in the control and in the limit test varied more than 1.5 units at the end of the test and chemical analyses revealed severe test item losses in the presence and absence of algae. This study is considered reliable with restrictions.
Justification for type of information:
α-Pinene is a bicyclic monounsaturated monoterpene and (-)-α-pinene is an enantiomeric form of α-pinene. Therefore, data on (-)-alpha pinene can be extrapolated to alpha-pinene. (see read-across justification document in section 13).
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH level in the control and in the limit test varied more than 1.5 units at the end of the test
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
pH level in the control and in the limit test varied more than 1.5 units at the end of the test
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2013-01-11
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.131 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
recalculated value
Details on results:
- A slight inhibition (38.5 %) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8 % at t = 48 h and 0 % at t = 72 h).
- Unusual cell shape: Microscopic examination of the shape of the algal cells every day did not show any difference between algae that had been growing at the limit test concentration and the algal cells in the control.
- Refer tables 6.1.5/2, 6.1.5/3 and 6.1.5/4 for more details

Regarding the test analyses results and taking into account the possibility that the organisms may not have been exposed to the test substance throughout the study, a more conservative exposure regime has been considered as a worst case scenario. A more conservative NOEC value for specific growth rate of 0.131 mg/L has been determined. This endpoint is supported by a High Accuracy QSAR (HA-QSAR) prediction using iSafeRat® with a worst-case ErC50 value of 0.31 mg/L
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Historical data (July 2012): ErC50 (72 h) = 0.96 mg/L
Reported statistics and error estimates:
- Statistical analysis was performed using software ToxRat® Professional.

ANALYTICAL RESULTS:

- Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day [biotic system: 0.905 mg/L at t = 0 h and between LOD (0.03 mg/L) and ( LOQ (0.1 mg/L) at t = 24h; abiotic system: 2.259 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24 h].

- These test item losses were considered to be associated to some degree with metabolism and/or adsorption effects of the test item by P. subcapitata, but given the significant test item losses also observed in the abiotic system, it would be more likely due to a photodegradation phenomenon or another abiotic degradation characteristic of the test item.

However, ecotoxicological results were recalculated in order to take into account losses of test item.

- Refer table 6.1.5/1 for more details

Table 6.1.5/1:Concentrations analysed (mg/L) of the test substance in test water - limit test: biotic and abiotic system

Treatment

Start (t = 0 h)

t = 24 h

t = 48 h

End (t = 72 h)

Biotic system*

Control

< LoQ

< LoQ

< LoQ

< LoQ

Limit concentration

0.905

LoD < C < LoQ#

LoD < C < LoQ#

LoD < C < LoQ#

Abiotic system

Limit concentration

2.259

LoD < C < LoQ#

0.108

0.104

* Preparation of samples for quantification of biotic system requested a transfer step in all-glass hemolysis tubes for centrifugation in contrast to abiotic system, which could explained the difference in concentration at t= 0 h found between biotic and abiotic system.

# At this time, the limit test concentration was below the LOQ (0.1 mg/L) but above the LOD (0.03 mg/L)

As substance concentration was between LoD and LoQ at T24h and T48h, there is proof that the algae must have been exposed to the test substance throughout the study (used for determination of the endpoint).

BIOLOGICAL RESULTS:

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10^4)

 

Replicate

Control

Limit concentration

T = 24h

1

2

3

4

5

6

4.0

3.6

4.0

2.8

2.0

4.0

1.2

2.0

2.4

2.0

1.2

1.2

Mean

Std. Dev.

3.4

0.83

1.7

0.53

T = 48h

1

2

3

4

5

6

22.8

24.4

16.4

22.4

24.8

20.0

16.8

17.6

18.0

14.0

11.2

18.4

Mean

Std. Dev.

21.8

3.15

16.0

2.83

T = 72h

1

2

3

4

5

6

52.8

52.4

55.2

49.2

63.2

67.2

56.0

54.0

58.0

59.2

54.0

56.4

Mean

Std. Dev.

56.7

7.00

56.3

2.10

Table 6.1.5/3: Yield (Y) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

Y

% I

Y

% I

Y

% I

Control

2.900

0

21.300

0

56.167

0

Test item

1.167

59.8

15.500

27.2

55.767

0.7

Table 6.1.5/4: Growth rate (G) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

G

% I

G

% I

G

% I

Control

1.887

0

1.883

0

1.575

0

Test item

1.161

38.5

1.725

8.4

1.574

0

Recalculation of endpoint-value:

The concentration measured in the abiotic sample are close to the limit of solubility of the tested substance (2.75 mg/L).

The lower concentration measured in the biotic sample may be explained by:

      - Loss due to the volatility and the additional manipulation of the samples prior to analysis due to the necessity to remove the algae by centrifugation. Test solution was transferred in centrifugation tubes to remove algae before analysis. The substance has a high Henry Constant and a few minutes would be enough to cause significant loss of material under these conditions.

      - Adsorption of the substance on algae but this does not imply substance loss from the test system. As abiotic samples do not undergo this step, we can consider that loss of substance occurred mostly in the biotic samples and that losses in the abiotic samples were much lower. Therefore the most appropriate concentration values closest to the actual exposure concentrations would be the abiotic sample concentrations.

The concentration of 2.259 mg/L is a better representation of the real exposure of organisms and the concentration of 0.905 mg/L represents the minimum exposure concentration (after significant volatility loss which occurred outside of the study during sample analysis).

The calculation of toxicity data based on the concentration of 0.905 mg/L is therefore considered as a worst-case value.

 To express a more conservative exposure of algae, the exposure concentration was calculated with the biotic samples. According to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, ENV/JM/MONO (2000)6, “ When the substance is detected but not quantified, it may be a good practice to use half of the limit of quantification.”At 24 and 48 h the substance is detected, the concentration is assumed to be 0.05 mg/L (half of the quantification limit, close to the detection limit). The exposure concentration over 48h was 0.131 mg/L, calculated as the mean geometric of 0.905, 0.05 and 0.05 mg/L (concentrations at 0, 24 and 48h).

  As inhibition is not significant at 48 h we can consider a NOEC of 0.131 mg/L.

Validity criteria fulfilled:
yes
Remarks:
control cultures (0-48 h): cell concentration increased by 44 folds; mean CV for section by section specific growth rate was 19 %; CV for average specific growth rate was 4.1 %
Conclusions:
The 48-h NOEC value of (-)-alpha pinene on Pseudokirchneriella subcapitata were estimated to be 0.131 mg/L after recalculation taking into account losses of test item during the experimental phase of the study.
This GLP study was performed according to OECD Guideline No. 201 and validity criteria were fulfilled.
Executive summary:

An algal toxicity study was performed on Pseudokirchneriella subcapitata with (-)-alpha-pinene in accordance with GLP and OECD Guideline OECD 201.

A preliminary test was conducted: algae were exposed to (-)-alpha pinene at nominal concentrations of 0.05, 0.10, 0.22, 0.46, 0.96 and 2.0 mg/L for 72 h. Except the first day at the higher test concentrations, no significant inhibition of the growth rate was recorded during the remainder of the test for all test concentrations.

In view of these results, a definitive test was conducted: algae were exposed to the test item at its solubility/saturation limit concentration (six replicate flasks per test concentration and control) for 72 h, in closed conditions under constant illumination and shaking at a temperature of 22.4 -23.5 °C. Measurement of biomass and growth inhibition was done by determination of cell densities at 24, 48 and 72 h using counting chamber. Samples taken from each treatment (biotic and abiotic medium) were analysed at the start and every day thereafter until the end of the test in order to determine maintenance of actual concentrations.

All the possible precautions described in the OECD document on testing difficult substance (OECD, 2000) and the OECD 201 Guideline were taken into account to conduct the study.

A slight inhibition (38.5%) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8% at t = 48 h and 0% at t = 72 h).

Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day [biotic system: 0.905 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24h; abiotic system: 2.259 mg/L at t = 0 h and between LOD (0.03 mg/L) and LOQ (0.1 mg/L) at t = 24 h]. The concentration measured in the abiotic sample are closed to the limit of solubility of the tested substance (2.75 mg/L).

To express a more conservative exposure of algae, the exposure concentration was calculated with the biotic samples. The concentration of 2.259 mg/L is a better representation of the real exposure of organisms and the concentration of 0.905 mg/L represents the minimum exposure concentration (after significant volatility loss which occurred outside of the study during sample analysis).

At 24 and 48 h the substance is detected, the concentration is assumed to be 0.05 mg/L (half of the quantification limit, close to the detection limit).

The exposure concentration over 48 h was 0.131 mg/L, calculated as the geometric mean of 0.905, 0.05 and 0.05 mg/L (concentrations at 0, 24 and 48h).

Therefore, the 48-h NOEC values of (-)-alpha pinene on Pseudokirchneriella subcapitata was estimated to be 0.131 mg/L.  

Validity criteria:

The cell density in the control cultures should have increased at least by a factor of 16 within exposure period (in this study a 44-fold increase was observed within 48 h).

The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35% (in this study 19% for 0-24h and 24-48h).

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (in this study 4.1% within 48h).

Therefore the validity criteria have been fulfilled and the study is considered valid over 48 h.

Description of key information

The 48h-NOErC for freshwater algae for (-)-alpha-pinene is 0.131 mg/L.


Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.131 mg/L

Additional information

An algal toxicity study was performed on Pseudokirchneriella subcapitata with (-)-alpha-pinene in accordance with GLP and OECD Guideline 201.

A slight inhibition (38.5 %) was recorded at 24 h at the limit test concentration in comparison to control cultures, but no significant inhibition of the growth rate was recorded during the remainder of the test (8 % at t = 48 h and 0 % at t = 72 h).

Chemical analyses revealed severe test item losses in the presence and absence of algae from the first day. These test item losses were more likely due to a photodegradation phenomenon or another abiotic degradation characteristic of the test item.

The NOEC values of (-)-alpha pinene on Pseudokirchneriella subcapitata was estimated to be 0.131 mg/L, respectively. After 72 h, no effect of the test item on Pseudokirchneriella subcapitata was observed at the solubility/saturation limit of the test item in the test water.

Thus the NOEC is valid for key value for chemical safety assessment.