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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 17 to 21, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 201
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH level in the control varied more than 1.5 units certainly due to the substantial algal growth in conjunction with closed conditions used in the test and this was not considered to have affected the integrity of the study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on July 05, 2012 / signed on January 11, 2013
Analytical monitoring:
yes
Details on sampling:
- Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with and without algae; the same biotic replicates were analysed at the start and the end of the test).
- Frequency of sampling: At the start (t=0 h) and the end of the test (t=72 h).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution was prepared by slow-stirring for the final test. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L of the test water was added. This is to use a maximum volume and to minimize head space whilst maintaining optimum surface contact between test item and the water. Then an excess of the test item (approximately 3 g) was carefully added directly to the surface of the test water. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. The stirring speed was kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 ± 2 hours of gentle stirring and a few minutes to settle (ca. 15 minutes), the saturated aqueous phase was taken out of the drain port. The first 100 mL were discarded and samples were taken from the following stock solution and chemically analyzed. Then the stock solution was directly diluted into flasks with test water and a fixed amount of inoculum to obtain the required test concentrations in the flasks and a cell density of 5 x 10^3 cells/mL per vessel.
The test sample of D-LIMONENE formed a clear colourless solution (except control vessels and non toxic concentrations of the test item, where solutions became greenish due to algal proliferation) and appeared to be completely soluble when mixed with test water and inoculum at the concentrations prepared, and remained unchanged during the 72 h of incubation.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.
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): Museum National d'Histoire Naturelle - 12, rue Buffon, Case No. 19 - 75005 PARIS, bred in the Laboratoires des pyrénées under standardised conditions according to the test guidelines.
- Stock culture: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.

ACCLIMATION
- Acclimation period: 2 to 4 days before the start of the test, cells from the algal stock culture were Inoculated in test water at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
None
Test temperature:
23.0-23.2 °C (average 23.1 °C)
pH:
At start of the test pH 8.08-8.16; at end of the test pH 7.68-10.21.
Although the pH level in the control varied more than 1.5 units at the end of the test this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and non-toxic concentrations of the test item was certainly due to the substantial algal growth in conjunction with closed conditions used in the test.
Dissolved oxygen:
None
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 0.2, 0.3, 0.5, 0.8, 1.3 and 2.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all glass flasks sealed with a fritted glass stopper.
- Type (delete if not applicable): Closed
- Aeration: Algal suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.
- Initial cells density: An initial cell density of 5 x 10^3 cells/mL using the exponentially growing preculture.
- No. of organisms per vessel: Volume of 50 mL of algal suspension per replicate
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the controls
- Additionally a replicate of each test concentration without algae was prepared in order to assess potential bioaccumulation and/or adsorption effects of the test Item by P. subcapitata during the test period and to determine maintenance of actual concentrations.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Original medium of OECD TG 201.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity was of 5474 lux (range: 5159-5947 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Range finding study: 0.5, 1.0 and 2.0 mg/L
- Results used to determine the conditions for the definitive study: After 72 h, no significant effect on algal growth was observed at the nominal exposure concentrations of 0.5 mg/L, while a significant algal growth inhibition was found at 1.0 mg/L up to the maximum solubility of test item in test water. Based on the results of a range finding test, test solutions used in the definitive test were prepared to obtain the following nominal concentrations (spaced by a factor of 1.58): 0.2, 0.3, 0.5, 0.8, 1.3 and 2.0 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 0.291-0.355 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.214 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL 0.193-0.244 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.174 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL 0.137-0.202 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.149 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL 0.110-0.169 mg/L
Results with reference substance (positive control):
In July 2012 (most recent test), the 72 h EC50 was 0.96 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72 h EC50: 0.60-1.03 mg/L).
Reported statistics and error estimates:
The evaluation of the effects was based on the average of measured exposure concentrations. The software ToxRat Professional was used for the determination of the 72 h ErCx and EyCx.

Chemical analyses

Chemical analyses revealed test item losses in the presence and absence of algae. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations in the biotic test, the results were based on the geometric means of measured exposure concentrations.

Nominal concentrations: 0.2, 0.3, 0.5, 0.8, 1.3 and 2.0 mg/L

Corresponding average exposure concentrations: -, 0.134, 0.189, 0.306, 0.536 and 0.938 mg/L

 

Table 6.1.5/1: Algal cell densities during the final test

 

Exposure duration

Replicate

Nominal concentration (mg/L)

Control

0.2

0.3

0.5

0.8

1.3

2.0

t = 24 h

1

2.0

1.2

2.0

0.8

1.6

0.4

0.4

2

2.0

4.0

2.8

1.6

1.6

0.8

0

3

3.6

1.2

1.2

2.8

0.4

0.4

0.8

4

4.0

NA

5

1.6

6

2.4

Mean

2.6

2.1

2.0

1.7

1.2

0.5

0.4

Std. Dev

0.97

1.62

0.80

1.01

0.69

0.23

0.40

t = 48 h

1

18.8

18.8

14.0

9.6

4.0

1.2

1.6

2

11.6

18.8

15.2

15.6

3.2

0.4

0.4

3

14.0

16.8

10.8

6.8

1.6

1.2

1.6

4

18.4

NA

5

11.6

6

17.6

Mean

15.3

18.1

13.3

10.7

2.9

0.9

1.2

Std. Dev

3.35

1.16

2.27

4.50

1.22

0.46

0.69

t = 72 h

1

58.0

58.4

48.0

32.0

7.2

1.2

0.8

2

56.4

66.8

68.8

59.2

7.6

0.8

0

3

60.0

52.0

53.2

27.6

2.4

1.6

0

4

62.4

NA

5

53.6

6

51.2

Mean

56.9

59.1

56.7

39.6

5.7

1.2

0.3

Std. Dev

4.12

7.42

10.82

17.12

2.89

0.40

0.46

Parameter

Growth rate (mg/L)

Yield (mg/L)

72 hour EC10

(95% confidential limits)

0.174

(0.137-0.202)

0.149

(0.110-0.169)

72 hour EC20

(95% confidential limits)

0.215

(0.180-0.241)

0.168

(0.137-0.187)

72 hour EC50

(95% confidential limits)

0.320

(0.291-0.355)

0.214

(0.193-0.244)

 

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

 

Validity criteria of the study

Cell density in controls: 114-fold increase within 72 hours

Coefficient of variation:

-The mean coefficient of variation for section by-section specific growth rates in the control cultures was of 25.4%.

-The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.5%.

Thus the validity criteria have been respected in the present study.

Validity criteria fulfilled:
yes
Conclusions:
Based on the average of measured exposure concentrations in the biotic test and under the experimental conditions, the 72-h EC50 for the parameters growth rate and yield were determined to be 0.320 mg/L and 0.214 mg/L, respectively. The 72-h EC10 for growth rate was 0.174 mg/L and for yield was 0.149 mg/L.
Executive summary:

In an algae growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item at the nominal concentrations of 0.2, 0.3, 0.5, 0.8, 1.3 and 2.0 mg/L for 72 h under static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and thus over several algae generations. The concentrations of the test item (with and without algae) were determined by chemical analyses at the start and at the 72 h incubation period.

 

Chemical analyses revealed test item losses in the presence and absence of algae. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations in the biotic test, the results were based on the geometric means of measured exposure concentrations. Corresponding average exposure concentrations: -, 0.134, 0.189, 0.306, 0.536 and 0.938 mg/L.

 

Based on the average of measured exposure concentrations in the biotic test and under the experimental conditions, the 72-h EC50 for the parameters growth rate and yield were determined to be 0.320 mg/L and 0.214 mg/L, respectively. The 72-h EC10 for growth rate was 0.174 mg/L and for yield was 0.149 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-23 May 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed according to OECD Guideline No. 201
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Density (20 °C): 0.842 g/mL
- Water solubility (20 °C): 4.0-5.7 mg/L
- Vapour pressure (298 °K): 200 Pa
Analytical monitoring:
yes
Details on sampling:
In order to verify the tested concentrations, two vials of each test concentration and control were harvested at the initiation of the test, every 24 h and at the termination of the test. In order to freeze the harvested samples, approx. 10-11 mL were discarded from the vials and the remaining approx. 30 mL were frozen immediately and stored at -20 ± 2.0 °C until dispatch for chemical analysis. Samples relevant for the calculation of the ECx, NOEC and LOEC concentrations were sent frozen to the analytical laboratory.
Duplicate samples were collected from each test concentration, both samples were analysed. As one of the B-samples was broken, it was decided to use only A-samples for data analysis. A good correlation was observed between the two replicate samples.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated stock solution (A) of 1 g/L was prepared by adding 2.65 mL (corresponding to 2.23 g) of the test item to 2.22 L test medium. The following procedure was used:
A 2-liter glass bottle with a magnet (total content 2.22 L) was filled with ISO medium withholding 5-10 mL of the total content. A pipette tip was saturated by filling it 3 times with the test item and 2.65 mL was carefully dosed to the middle of the water phase. The bottle was rapidly filled totally with ISO medium and sealed tightly with a screw cap with PTFE packing. The bottle was covered with black cloth and left for gentle stirring, approximately 350 rpm, at room temperature for 24 ± 2 h. The stirring was stopped and approximately 1750 mL of the mid fraction syphoned off using a silicone tube connected to a glass tube with narrow diameter. The first 100 mL of the mid fraction was discarded and the rest transferred to a new glass bottle with as little headspace as possible. The mid fraction (stock solution B) was carefully mixed well without shaking. pH was measured to be 7.9 and no adjustment was therefore necessary. The test solutions were prepared individually by immediately diluting stock solution B in test medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Clone: NIVA, CHL 1
- Source (laboratory, culture collection): Cultured at test facility under the test conditions prior to the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
22.3 ± 0.1 °C
pH:
At the start of the test, pH was measured to be 7.9 and at the termination it was 7.9-9.4.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 7, 10, 16, 24, 35, 53 and 80 % of a saturated solution of the test item in test medium.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test was carried out in 42-mL glass vials sealed with PTFE-coated screw caps each containing 41 mL of test solution consisting of algal growth medium, algae and test item.
- Initial cells density: At test start, the cell density was approx. 2500 cells/mL, measured in the control.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): six controls (vials with algae but no test item); a blank of each concentration (vials with test item but no algae)
- Furthermore, six vials of the control and six vials of each test concentration without algae were made to enable harvesting of duplicate samples for chemical analysis at each sampling point.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium according to ISO 8692

OTHER TEST CONDITIONS
- Light intensity and quality: Constant illumination from a panel of fluorescent light with an intensity of approx. 60-120 μmol/m2/sec.

EFFECT PARAMETERS MEASURED
- At the beginning of the test and after 24, 48 and 72 ± 2 h of incubation, the algal growth was followed in the triplicate test vials, the blanks and the six controls by measuring the fluorescence.
- For the measuring range used in the test, the correlation between cell counts, using a Beckman Coulter Multisizer, and in-vivo fluorescence, using a Turner TD-700 Laboratory Fluoro-meter, is validated twice a year.
- The identity and normal appearance of Pseudokirchneriella subcapitata in the control was confirmed at the end of the test by microscopy.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 0.13-0.16 mg/L
Duration:
48 h
Dose descriptor:
EC20
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 0.16-0.19 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 0.24-0.27 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The NOEC values determined on the basis of the yield by Dunnett’s procedure shows a significant inhibition at the lowest tested concentration (7 % saturated solution of the test item corresponding to 0.05 mg/L test item) and the values are thus given as <7 % and <0.05 mg/L, respectively.
Results with reference substance (positive control):
A test with the reference substance potassium dichromate was performed under the same conditions as for the test item in order to verify the sensitivity of the algae. The reference test was performed at the following concentrations: 0 (control), 0.10, 0.20, 0.40, 0.70, 1.0, 1.4 and 2.0 mg/L.
- The EC50 value of 0.91 (0.90-0.92) mg/L showed that the sensitivity of the algae was not significantly different from the results obtained in an international ring test in 1981 (EC50 = 1.19 (0.65-1.73) mg/L).
Reported statistics and error estimates:
Growth inhibition and growth were calculated for each test concentration relative to the control without addition of test item. The concentrations inhibiting the growth 10, 20 and 50 % were calculated for two different response variables, the growth rate (ErC10, ErC20 and ErC50) and the yield (EyC10, EyC20 and EyC50) by use of the computer program TOXEDO. The NOEC value was determined by use of the computer program Dunnett’s procedure as the highest tested concentration, at which no significant inhibition was observed on growth rate compared with the control. As all tested concentrations caused significant inhibition on yield, no NOEC value could be determined on yield.

Table 6.1.5/1: Results of the chemical analyses of subsamples from the test solutions without algae

Sampling time

 

t = 0 hours (mg/L)

 

t = 24 hours (mg/L)

 

t = 48 hours (mg/L)

 

t = 72 hours (mg/L)

 

Geometric mean (mg/L)

 

Control

 

<0.010

-

-

<0.010

<0.010

7 %

0.08

0.12

0.06

<0.010

0.05

10 %

0.13

0.15

0.12

<0.010

0.08

16 %

0.20

0.29

0.17

<0.010

0.12

24 %

0.36

0.31

0.24

0.010

0.17

 

The results showed a major decrease in the actual measured test concentration from the 48 h sample to the 72 h sample. However, in the same period, no direct correlation was seen between the drop in the d-Limonene concentration and the algal growth.

Validation

The control growth rate exceeded 1.4 per day /2/ (growth rate: 0.072 per h ~ 1.7 per day).

The variation coefficient of the control growth rates did not exceed 5 % (variation coefficient: 1.0 %).

The control pH did not increase more than 1.5 during the test (a pH increase of 1.5).

The mean value of the variation coefficient of the growth rates in the control replicates, calculated in sections of 24 h during the 72 h test period, did not exceed 35 % (mean variation coefficient: 11.4 %).

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC50 for the parameters growth rate and yield were determined to be 0.05 mg/L and <0.05 mg/L, respectively. The EC10, EC20 and EC50 for growth rate was 0.09, 0.11 and 0.15 mg/L, respectively and EC10, EC20 and EC50 for yield was 0.05, 0.06 and 0.09 mg/L, respectively, based on the geometric means of measured exposure concentrations.
Executive summary:

In an algae growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to d-limonene at the nominal concentrations of 7, 10, 16, 24, 35, 53 and 80% of a saturated solution of the test item in test medium for 72 h. The growth of the algae was followed during 72 h by measuring the fluorescence at the beginning of the test and every 24 h. The concentrations of the test item were determined by chemical analyses at 0, 24, 48 and 72 h incubation period.

   

The NOEC values determined on the basis of the yield by Dunnett’s procedure shows a significant inhibition at the lowest tested concentration (7% saturated solution of the test item corresponding to 0.05 mg/L of test item) and the values are thus given as <7 % and <0.05 mg/L, respectively.

Growth rate:

72-h NOEC: 0.05 mg/L; EC10: 0.09 mg/L (95% Cl 0.08-0.09 mg/L); EC20: 0.11 mg/L (95% Cl 0.10-0.11 mg/L); EC50: 0.15 mg/L (95% Cl 0.15-0.16 mg/L)

Yield:

72-h NOEC: <0.05 mg/L; EC10: 0.05 mg/L (95% Cl 0.04-0.06 mg/L); EC20: 0.06 mg/L (95% Cl 0.05-0.07 mg/L); EC50: 0.09 mg/L (95% Cl 0.08-0.11 mg/L)

Therefore, the 72-h EC50 for parameters growth rate and yield were determined to be 0.05 mg/L and <0.05 mg/L, respectively.

The EC10, EC20 and EC50 for growth rate was 0.09, 0.11 and 0.15 mg/L, respectively and EC10, EC20 and EC50 for yield was 0.05, 0.06 and 0.09 mg/L, respectively, based on the geometric means of measured exposure concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2015-12-07 to 2015-12-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR value. The substance falls into applicability domains of the model QSAR.
Justification for type of information:
QSAR prediction
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
QSAR model
Principles of method if other than guideline:
The growth inhibition of algae was determined using validated QSAR models for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR models are based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 and 31 chemicals derived from 72-hour NOECr test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
not applicable
Test organisms (species):
other: algae spp.
Details on test organisms:
not applicable
Test type:
other: QSAR model
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
72h-ErC50 and 72h-NOECr
Post exposure observation period:
not applicable
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The QSAR is based on data from studies performed at between 20 - 25 °C (depending on the species considered).
pH:
The QSAR is based on data from studies performed at acceptable pH between 6 - 9.
Dissolved oxygen:
The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).
Salinity:
The QSAR is based on data from studies performed on freshwater species.
Nominal and measured concentrations:
The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.
Details on test conditions:
not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.5 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: [0.42-0.60]
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: [0.14-0.24]
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
72h-ErC50 = 0.50 mg/L with 95% CL [0.42-0.60]
72h-NOECr = 0.18 mg/L with 95% CL [0.14-0.24]
QSAR statistical parameters are given in the QMRF and QPRF

no data

Validity criteria fulfilled:
yes
Remarks:
The substance falls into applicability domains of the model QSAR.
Conclusions:
72h-ErC50 for d-limonene = 0.50 mg test item/L based on growth rate inhibition with 95%-Confidence Limit of 0.42-0.60 mg test item/L.
72h-NOECr for d-limonene = 0.18 mg test item/L based on growth rate inhibition with 95%-Confidence Limit of 0.14-0.24 mg test item/L.
Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question (MOA 1, non-polar narcosis). The QSAR models are based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 and 31 chemicals derived from 72-hour NOECr test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of d-limonene has been investigated using a QSAR model that predicts growth rate inhibition of algae in an OECD 201 study. d-Limonene falls within the applicability domain of the model as demonstrated in the QPRF.

The 72-h ErC50 of d-limonene was predicted as 0.50 mg/L based on growth rate inhibition. 

The 72-h NOECr of d-limonene was predicted as 0.18 mg/L based on growth rate inhibition. 

This toxicity study is acceptable and can be used for that endpoint.

 

Description of key information

The substance exhibits an EC50 for freshwater algae of 0.32 mg/L and an EC10 of 0.174 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.32 mg/L
EC10 or NOEC for freshwater algae:
0.174 mg/L

Additional information

Two experimental studies are available and are supported by valid QSAR data.

In an algae growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae speciesPseudokirchneriella subcapitatawas exposed to the test item at the nominal concentrations of 0.2, 0.3, 0.5, 0.8, 1.3 and 2.0 mg/Lfor 72 h under static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and thus over several algae generations. The concentrations of the test item (with and without algae) were determined by chemical analyses at the start and at the 72 h incubation period.

In an algae growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitatawas exposed to d-limonene at the nominal concentrations of 7, 10, 16, 24, 35, 53 and 80% of a saturated solution of the test item in test mediumfor 72 h. The growth of the algae was followed during 72 h by measuring the fluorescence at the beginning of the test and every 24 h. The concentrations of the test item were determined by chemical analyses at 0, 24, 48 and 72 h incubation period.