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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
Study period:
20 May 2011 to 23 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
1996
Deviations:
yes
Remarks:
In accordance with the OPPTS Test Guideline, test duration would be 96 hours. However, due to the instability of the test item in the test water the test duration was 72 hours.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: VE00106679
Purity: 91.60% (sum of the 2 isomers)
Physical Form: Colourless to pale yellow liquid
Expiration Date: 10-Jul-2012
Storage Conditions (as provided by the Sponsor): At room temperature, protect against light.
Storage Conditions (as handled at Harlan Laboratories): At room temperature at about 20 °C, away from direct sunlight.
Analytical monitoring:
yes
Details on sampling:
For preparation of the WAFs, individual dispersions of the test item were prepared:

Loading Rate (mg/L) Preparation
3.2 7.4298 mg test item in 2320 mL test water
10 23.2388 mg test item in 2320 mL test water
32 74.25 mg test item in 2320 mL test water
100 233.15 mg test item in 2330 mL test water

The dispersions were stirred for 96 hours at room temperature in the dark to dissolve a maximum amount of the different test item components in the dispersions. The stirring period of 96 hours was considered appropriate to dissolve a maximum amount of the hardly soluble test item components in the test water and was based on the results of a stirring experiment (non-GLP, see APPENDIX IV). After stirring, the dispersions were left to settle for about 3 hours to separate undissolved test material from the test water (as far as possible) and the middle layers were drawn off with a Teflon® tube for filtration. The middle layers were filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) after preconditioning the filter material with about 300 mL of test media to avoid test item loss in the filter. The negative pressure of the filtration unit was reduced as much as possible to avoid loss of volatile components of the test item during filtration. The undiluted filtrates were tested as WAFs.
Due to technical reasons, the WAFs with the lowest loading rates of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.
The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The test media were prepared just before the start of the exposure.
Vehicle:
no
Details on test solutions:
Since the test item was determined to be volatile, glass stoppered 50 mL Erlenmeyer flasks were used (closed system) completely filled with about 60 mL of test medium to minimize the air- space in the flasks and to avoid potential loss of test item. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.
The test flasks were incubated in a temperature-controlled water bath at a temperature of 21 °C and illuminated by fluorescent tubes (Philips TLD 36W/840 – cool white type), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 5100 Lux (range: 4650 to 5560 Lux, measured at nine places in the experimental area). The light intensity was within a ±15%-deviation from the average light intensity as recommended by the guideline.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
In accordance with the OPPTS Test Guideline, test duration would be 96 hours. However, due to the instability of the test item in the test water the test duration was 72 hours.
Test temperature:
The water temperature during the test was maintained at 21 °C
pH:
At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values of 8.2 to 8.6 were measured.
Nominal and measured concentrations:
Based on non-GLP dose-range finding, since the test item is a multi-component substance, water accommodated fractions (WAFs) with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control was tested in parallel (test water without test item).
The enlarged spacing factor of 3.2 between the test concentrations was chosen since a rather flat dose-response relationship was expected applying the WAF-approach.
At the start of the test, the measured test item concentrations in the test media with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.048, 0.20, 0.71, 1.7, 2.0 and 3.3 mg/L, respectively.
The 72-h mean measured concentrations were 0.026, 0.16, 0.56, 1.4, 1.6 and 2.7mg/L, respectively.
The 72-h mean measured concentrations corrected for algal uptake were 0.028, 0.18, 0.63, 1.50, 1.82 and 3.01 mg/L, respectively.
Details on test conditions:
The test design included three replicates per test concentration and six replicates of the control.
In order to evaluate the influence of the presence of light and algae on the stability of the test item, additional flasks containing the test medium of the 32 mg/L-WAF (also used for the toxicity testing) were set up in a closed system. Three different incubation conditions, “Light with algae”, “Light without algae”, and “Darkness without algae”, were applied.
The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for algal uptake - 95% CI 2.2-3.2
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for algal uptake - 95% CI 0.88-1.5
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for algal uptake - 95% CI 0.49-1.2
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.028 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for algal uptake
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: corrected for algal uptake
Details on results:
In the full study report the 48-h and 72-h biological results are reported based on i) loading rates of the test item, ii) mean measured concentrations of the test item and iii) mean measured concentrations corrected for algal uptake. Only the 72-h effects results based on mean measured concentrations corrected for algal uptake are presented in the above effect concentrations table. These are considered the most appropriate for regulatory purposes for the following reasons:
• 72-h effect results are required for classification purposes
• Given that initial measured concentrations were significantly lower than the WAF loading rates and that test item concentrations decreased over the 72 hour test duration to 43 to 70% of the initial concentrations, it is considered more appropriate to base effect results on mean measured concentrations as opposed to loading rates. Further, although effect rates based on WAF loading rates can be used directly for classification this only applies for multi-constituent substances where there are differences in partitioning behaviour and water solubility between constituents. In contrast, KARMALONE is a mixture of two isomers which are expected to have similar solubility. This was observed in the water solubility study (see IUCLID endpoint 4.8) where at the end of the experiment, it was seen that the ratio between the two peaks of KARMALONE remained constant even in saturated solutions.
• In a parallel stability study, the main loss of test substance in the test water was attributed to adsorption to algal cells which accounted for about 25% of the loss over 72 hours. Since the algal cells are, in effect, exposed to this complimentary fraction of test substance, this loss was considered in the calculation of the study endpoints (i.e. the effect results were based on the 72-h mean measured concentrations corrected for algal uptake.)

For comparison, the corresponding effect results based on the time-weighted mean measured concentrations (direct analysis) are shown below. These represent a worst-case analysis of the data because they are based only on the test item dissolved in the test media. However, the results are only slightly lower than those based on mean measured concentrations (corrected for algal uptake) with the effect values relevant for classification and labelling (e.g. EC50 and EC10) falling in the same classification band.
EC50 72 hours: 2.3 mg/L
EC20 72 hours: 1.2 mg/L
EC10 72 hours: 0.82 mg/L
NOEC 72 hours: 0.026 mg/L
LOEC 72 hours: 0.16 mg/L

Effects based on yield were also reported (see attached full study report). However, the preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 2.6 mg/L and 0.88 mg/L respectively.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in April 2011 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.3 mg/L (Harlan Laboratories Study D25712), range of the 72-hour EC50 for the growth rate from 2000 to 2011: 0.71-1.7 mg/L).
Reported statistics and error estimates:
The test item had a statistically significant inhibitory effect on the growth rate μ of the algae after the test periods of 48 and 72 hours at the loading rate of 1.0 mg/L, corresponding to the mean measured concentrations of 0.18 (0.19) (48 hours) and 0.16 (0.18) mg/L (72 hours), and at the higher test concentrations (results of Welch t-test, one-sided smaller, α = 0.05). Thus, the 48- and 72-hour NOECs based on the growth rate μ were determined to be at the loading rate of 0.32 mg/L, corresponding to the mean measured concentrations of 0.040 (0.042) and 0.026 (0.028) mg/L, respectively, since up to and including these test concentrations the growth rate of the algae was not statistically significantly inhibited compared to the control.

The inhibitory effect on the yield Y of the algae was statistically significant at the loading rate of 1.0 mg/L (mean measured test concentration of 0.18 (0.19) mg/L) after 48 hours and at the next lower loading rate of 0.32 mg/L (mean measured concentration of 0.026 (0.028) mg/L) after 72 hours. However, after 72 hours of test duration, the inhibition of yield at the mean measured concentration of 0.026 (0.028) mg/L was only 8.9%. Since in accordance with the test guidelines an effect of 10 to 20% seems appropriate to represent the NOEC, the 48- and 72-hour NOECs for yield were determined to be at the loading rate of 0.32 mg/L, corresponding to the mean measured concentrations of 0.040 (0.042) and 0.026 (0.028) mg/L, respectively. No biologically relevant toxic effects of the test item on the yield of the algae was observed at these test concentrations compared to the control.
Validity criteria fulfilled:
yes
Conclusions:
The biological results based on time-weighted mean measured concentrations (corrected for algal uptake) were as follows:
EC50 72 hours: 2.6 mg/L
EC20 72 hours: 1.3 mg/L
EC10 72 hours: 0.88 mg/L
NOEC 72 hours: 0.028 mg/L
LOEC 72 hours: 0.18 mg/L

The above results are considered the most appropriate for regulatory purposes as algal cells are, in effect, exposed to the test item that is both adsorped to algal cells and dissolved in the test media.

For comparison, the corresponding effect results based on the time-weighted mean measured concentrations (direct analysis) are shown below. These represent a worst-case analysis as they are based only on the test item dissolved in the test media. However, ithe results are only slightly lower than those based on mean measured concentrations (corrected for algal uptake) with the effect values relevant for classification and labelling (e.g. EC50 and EC10) falling in the same classification band.
EC50 72 hours: 2.3 mg/L
EC20 72 hours: 1.2 mg/L
EC10 72 hours: 0.82 mg/L
NOEC 72 hours: 0.026 mg/L
LOEC 72 hours: 0.16 mg/L

Executive summary:

The influence of the test item Cosmone on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3, as well as according to the OPPTS Guideline No. 850.5400 (Public Draft, April 1996).

In order to assess the toxicity of the multi-component test item Cosmone to algae, and following a non-GLP range-finding test, water accommodated fractions (WAFs) with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures,2000.

 

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 96 hours to dissolve a maximum amount of the different components of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.45 µm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAFs with the lowest loading rates of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.

 

As the test item is a volatile substance, the test was performed using Erlenmeyer flasks completely filled with test medium and tightly sealed with glass stoppers to avoid loss of test item. Three replicates per treatment with nominal 10’000 algal cells/mL were exposed to the test item during 72 hours under continuous lighting at 21 °C. The algal biomass in the samples was determined by fluorescence measurement.

At the end of the test, the shape and size of the algal cells were visually inspected, and, no adverse effect was observed.

 

In order to evaluate the influence of the presence of light and algae on the stability of the test item, additional flasks containing the test medium of the 32 mg/L-WAF (also used for the toxicity testing) were set up in a closed system. Three different incubation conditions, “Light with algae”, “Light without algae”, and “Darkness without algae”, wereapplied.

 

All samples were stored frozen, thawed prior to analysis and the algal cells separated from the aqueous phase by centrifugation. Analysis was performed using HPLC with UV-detection.

 

At the start of the test, the measured test item concentrations in the test media with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.048, 0.20, 0.71, 1.7, 2.0 and 3.3 mg/L, respectively. During the test period of 72 hours, a decrease of the measured test item concentrations in the test media occurred. After 48 hours and 72 hours of test duration, test item concentrations had decreased to 43 to 79% and to 43 to 70% of the values measured at test start, respectively.

In the parallel stability study, the main loss of test substance in the test water was attributed to adsorption to algal cells which accounted for about 5% of the starting concentration over the period of 24 hours, 20% over 48 hours, and, 25% over 72 hours. This loss was considered in the calculation of the study endpoints, as the algal cells are, in effect, exposed to this complimentary fraction of test substance. Losses due to aqueous photolysis were < 10%.

 

Incubation Conditions

 

Day 0

Day 1

Day 2

Day 3

 

Light + Algae

Concn. (mg/L)

2.01

1.86

1.60

1.36

% of Day 0

100

92.5

79.6

67.7

Light without Algae

Concn. (mg/L)

2.01

2.02

2.00

1.86

% of Day 0

100

100.5

99.5

92.5

Darkness without Algae

Concn. (mg/L)

2.01

2.12

1.92

2.02

% of Day 0

100

105.5

95.5

100.5

 

 

The 48- and 72-hours mean measured concentrations were as follows:

 

 

 

 

 

Loading rate (mg/L)

Mean measured concentrations (time-weighted means)

48 hours

72 hours

 

Mean measured concentrations

(mg/L)

Mean measured concentrations corrected for algal uptake+

(mg/L)

 

Mean measured concentrations

(mg/L)

Mean measured concentrations corrected for algal uptake+

(mg/L)

0.32*

0.040

0.042

0.026

0.028

1.0

0.18

0.19

0.16

0.18

3.2

0.62

0.67

0.56

0.63

10

1.5

1.59

1.4

1.50

32

1.8

1.93

1.6

1.82

100

2.8

3.03

2.7

3.01

* at this loading rate the measured concentration after 48 hours and at the end of the test was < LOQ; the

½ LOQ (0.0209 mg/L) was used as 48 and 72-hour-concentration to calculate the time-weighted means.

+Mean time-weighted concentrations calculated from sample injections and corrected for fraction of test substance adsorbed by algal cells (5% for 24-hour values, 20% for 48-hour values, 25% for 72-hour

values).

The biological results based on time-weighted mean measured concentrations were as follows:

 

 

after 48 h

after 72 h

Parameter

 

Growth rate

Yield

Growth rate

Yield

EC50

(mg/L) (95% CI)

Direct Analysis

3.2* (2.7 - 4.2*)

1.5

(1.3 – 1.7)

2.3

(2.0 - 2.8*)

0.49

(0.39 - 0.59)

Corrected for Algal Uptake

3.4* (3.0 – 4.6*)

1.6

(1.4 – 1.8)

2.6

(2.2 – 3.2*)

0.55

(0.44 – 0.66)

EC20

(mg/L) (95% CI)

Direct Analysis

1.6

(1.3 - 1.8)

0.58

(0.40 - 0.73)

1.2

(0.82 - 1.4)

0.13

(0.080 - 0.18)

Corrected for Algal Uptake

1.7

(1.4 – 1.9)

0.62

(0.43 – 0.78)

1.3

(0.88 – 1.5)

0.15

(0.093 – 0.21)

EC10

(mg/L) (95% CI)

Direct Analysis

1.1

(0.74 - 1.4)

0.35

(0.21 - 0.48)

0.82

(0.46 - 1.1)

0.070

(0.040 - 0.10)

Corrected for Algal Uptake

1.2

(0.77 – 1.5)

0.38

(0.23 – 0.52)

0.88

(0.49 – 1.2)

0.076

(0.040 – 0.12)

NOEC

(mg/L)

Direct Analysis

0.040

0.040

0.026

0.026

Corrected for Algal Uptake

0.042

0.042

0.028

0.028

LOEC

(mg/L)

Direct Analysis

0.18

0.18

0.16

0.16

Corrected for Algal Uptake

0.19

0.19

0.18

0.18

95%CI:    95% confidenceinterval

*:              extrapolatedvalue

 

 

The biological results based on loading rates of the test item were as follows:

 

 

after 48 h

after 72 h

Parameter

Growth rate

Yield

Growth rate

Yield

EC50(mg/L)

156*

19

67

3.8

95% CI

99 – 331*

15 – 24

45 – 123*

3.1 – 4.6

EC20(mg/L)

18

2.6

8.5

0.65

95% CI

10 - 26

1.7 – 3.6

4.1 - 14

0.44 - 0.87

EC10(mg/L)

5.7

0.92

2.9

0.26

95% CI

2.2 – 9.8

0.50 – 1.5

0.88 – 5.6

0.15 - 0.38

NOEC (mg/L)

0.32

0.32

0.32

0.32

LOEC (mg/L)

1.0

1.0

1.0

1.0

95%CI:          95% confidenceinterval

*:                    extrapolatedvalue

Description of key information

The influence of the test item Cosmone on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3, as well as according to the OPPTS Guideline No. 850.5400 (Public Draft, April 1996).

ErC50(72hrs) = 2.6 mg/L (95% confident interval between 2.2 and 3.2 mg/L)

ErC10(72hrs) = 0.88 mg/L (95% confident interval between 0.49 and 1.2 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:
2.6 mg/L
EC10 or NOEC for freshwater algae:
0.88 mg/L

Additional information

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 2.6 mg/L and 0.88 mg/L respectively.

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), for rapidly degradable substances an EC10 of 0.88 mg/L corresponds to Hazardous to the Aquatic Environment Chronic 3 classification.

However, Chronic 1 classification should apply based on daphnia EC50 below 1mg/L and log Kow => 4.