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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:
weight of evidence
Study period:
March 2018 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted March 23, 2006, corrected July 28, 2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
August 24, 2009
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Qualifier:
according to
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: Ginger extract (volatile)
Synonym: Ginger CO2-se extract, type no. 014.001
Batch No.: 461108
Known Constituents Content:
Volatile oils: 88.9 %
Gingerol: 2.2 %
Shogaol: 0.42 %

Analysis Date: August 02, 2016
Type: UVCB
Aggregate State at Room Temperature: Liquid
Colour: Yellow
Density: 0.8838 g/cm³ (20°C)
Retest Date: July 2021
Storage Conditions at Test Facility:
At 20 +/- 5 °C, in the dark
Analytical monitoring:
yes
Remarks:
GC-FID peak area
Details on sampling:
Sampling:
The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.

Storage:
All samples were extracted with an organic solvent stand-by immediately after sampling. The extracts were stored in a refrigerator (4 ± 4°C), protected from light until analysis was performed. Afterwards the samples were again stored refrigerated and will be kept stored up to the date of the final report.

Analyses:
The dosage of the test item Ginger extract (volatile) was analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples, from both sampling times (0 and 72 hours).
Vehicle:
no
Remarks:
Water accomodated fractions were used
Details on test solutions:
Dosage of Test Item:
A defined amount of the test item was added directly to the test water for each test loading rate and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item loading rate of 100 mg test item/L was prepared by mixing 105.2 mg test item into 1052 mL test water, for the test item loading rate of 32 mg test item/L, 33.6 mg test item were mixed into 1050 mL test water, for the loading rate of 10 mg test item/L, 10.5 mg were mixed into 1050 mL test water. The loading rate of 3.2 mg test item/L was prepared by mixing 18.6 mg into 5800 mL test water and for 1.0 mg test item/L, 11.4 mg were mixed into 11400 mL test water. After cessation of mixing and a following period (0.5 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test loading rate. The test media were prepared just before introduction of the algae (= start of the test).

Appearance of the Test Item in Test Medium: There were no remarkable observations
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Origin: The algae were supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: Algae cells were taken from an exponentially growing pre-culture 4 days prior
- Culturing media and conditions (same as test or not): pre-culture medium and test medium are the same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3
Test temperature:
22.1 to 23.2 °C
pH:
Test start: 8.2 to 8.3
Test end: 8.6 to 9.9

The pH in the control increased by more than 1.5 units. However, this does not invalidate the tests since the validity criterion was met.
Nominal and measured concentrations:
Water accommodated Fractions (WAF) nominal loading rates: Control, 1.0, 3.2, 10, 32 and 100 mg/L

Dose dependent presence of test item based on peak area was shown on t=0h and t=72h (<= 54% of initial).
Details on test conditions:
TEST CONDITIONS:
- Type and Size: Erlenmeyer flasks of 50 mL volume containing as much test medium as possible, i.e. at least 50 mL in order to reduce the remaining head space to a technical possible minimum of some mL, kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- Control end cells density: 85.202 [10000/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Light Regime: Continuous illumination
- Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 6953 lux (range: 6100 to 7660 lux)

GROWTH MEDIUM
- Standard medium used: yes - OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
- Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Non-GLP pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
40.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 37.0 - 44.0 mg test item/L)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
13.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 10.8 - 16.0 mg test item/L)
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes

- Observation of abnormalities: no -The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test loading rate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test loading rate, the highest loading rate.

No remarkable observations, clear test medium
The pH in the control increased by more than 1.5 units. However, this does not invalidate the tests since the validity criterion was met.

Results with reference substance (positive control):
Results with reference substance are valid. Historical ranges not included
72h-ErC50: 1.02 mg/L (95% C.I. 0.984-1.05 mg/L)
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErL50 and the 72 hours EyL50, the corresponding EL20 and EL10 values and where possible their 95 %-confidence limits were calculated by Probit analysis for yield and by Weibull analysis for growth rate.
For the determination of the 72 hours LOEL and the 72 hours NOEL, the calculated growth rates and yields at each test loading rate were tested for significant differences compared to the control values by Bonferroni-Welch t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical Results:

The qualitative analysis of the test item Ginger extract (volatile) in the test samples was performed using gas chromatography with flame ionization detection. From the analytical data it was shown that WAFs were prepared correctly based on increase in peak areas with loading rate. At test end the blank corrected total peak areas could not be adequately quantified for the two lowest loading rates. However, peak patterns could still be differentiated compared to the control, therefore confirming the presence of test item components at test end. For the three higher loading rates up to 54% of initial peak areas was observed at test end.

Yield y and Percentage Inhibition of y during the Test Period

 

Nominal loading rates [mg test item/L]

Yields y [10000 cells/mL] and % inhibition of y

 

 

0-24 hrs

0-48 hrs

0-72 hrs

 

y

%

y

%

y

%

control

1.933

-

14.950

-

84.702

-

1.0

1.560

19.3

14.121

5.5

83.476

1.4

3.2

0.316

 83.7*

13.328

10.9*

84.053

       0.8

10

1.187

38.6*

4.893

67.3*

38.994

54.0*

32

0.000

100.0*

0.330

97.8*

14.626

82.7*

100

0.067

96.5*

0.000

100.0*

0.000

100.0*

 

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control
(tested with Bonferroni-Welch t-test (24h and 72h) and Williams t-test (48h),a = 0.05, one-sided)

 

Growth Rates μ and Percentage Inhibition of μ during the Test Period

Nominal loading rates [mg test item/L]

Growth rates μ [1/day] and % inhibition of μ

 

 

0-24 hrs

0-48 hrs

0-72 hrs

 

μ

%

μ

%

μ

%

control

1.552

-

1.714

-

1.713

-

1.0

1.415

8.8

1.688

1.6

1.707

0.3

3.2

0.468

69.8*

1.659

3.2

1.709

0.2

10

1.210

22.0*

1.188

30.7*

1.454

15.1*

32

0.000

100.0*

0.194

88.7*

1.135

33.7*

100

0.115

92.6*

0.000

100.0*

0.000

100.0*


negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control
(tested with Bonferroni-Welch t-test,a = 0.05, one-sided)

Validity criteria fulfilled:
yes
Remarks:
In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%
Conclusions:
The ErL50, ErL10 and NOELR were 40.4, 13.2 and 3.2 mg test item /L respectively.
Executive summary:

Algae toxicity was assessed in an OECDTG 201 static concentration-response GLP study with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per treatment group were exposed to WAFs prepared at loading rates of 100, 32, 10, 3.2 and 1.0 mg Ginger extract (volatile) per litre in closed vessels.The qualitative analysis of the test item Ginger extract (volatile) in the test samples was performed using gas chromatography with flame ionization detection. From the analytical data it was shown that WAFs were prepared correctly based on increase in peak areas with loading rate. Presence of test item (up to 54% of initial) at test end was also shown for the three highest loading rates. All reported results refer to nominal loading rates.The 72-hour ErL50, ErL10 and NOELR were 40.4, 13.2 and 3.2 mg test item /L respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 2018 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted March 23, 2006, corrected July 28, 2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
August 24, 2009
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Qualifier:
according to
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: Ginger extract (non-volatile)
Synonym: Ginger Hot Flavor CO2-to extract, type no. 014.008
Batch No.: 861213
Analysis Date: July 20, 2016
Type: UVCB
Aggregate State at Room Temperature: Liquid
Colour: Brown
Retest Date: July 2019
Storage Conditions at Test Facility: At 20 +/- 5 °C, in the dark

Known Constituents Content:
Gingerol: 44.7 %
Shogaol: 3.4 %,
according to certificate of analysis
Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
Sampling:
The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated during test period under the same conditions as in the actual test and from these test media duplicate samples were taken at the end of the test period. The collecting of samples after 72 hours from the actual test itself was not possible, since the test media volumes in the test were too small for the analytical requirements. The samples remained undiluted until analysis.

Storage:
All samples were stored in a fridge (4°C ± 4°C), protected from light until analysis was performed. Afterwards the samples were again stored cooled (4°C ± 4°C) and will be kept stored up to the date of the final report..

Analyses:
The dissolved fraction of the test item Ginger extract (non-volatile) was analysed in the duplicate test media samples from all test concentrations and the control samples from both sampling times (0 and 72 hours).
Vehicle:
no
Details on test solutions:
Dosage of Test Item:
A defined amount of the test item was added directly to the test water for each test loading rate and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item loading rate of 100 mg test item/L was prepared by mixing 105.9 mg test item into 1059 mL test water, for the test item loading rate of 32 mg test item/L, 33.5 mg test item were mixed into 1047 mL test water, for the loading rate of 10 mg test item/L, 24.4 mg were mixed into 2440 mL test water. The loading rate of 3.2 mg test item/L was prepared by mixing 18.6 mg into 5800 mL test water and for 1.0 mg test item/L, 11.4 mg were mixed into 11400 mL test water. After cessation of mixing and a following period of half an hour of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test loading rate.The test media were prepared just before introduction of the algae (= start of the test).

Appearance of the Test Item in Test Medium: The visual appearance of the test item in test water was determined daily in all test item loading rates. At test start coloration was observed at the loading rates of 100, 32 and 10 mg test item/L. The coloration was not visible after 24 h at the loading rate of 10 mg test item/L and after 48 h not at the loading rate of 32 mg/L anymore. A slight coloration remained at the highest loading rate after 72 h
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Origin: The algae were supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: Algae cells were taken from an exponentially growing pre-culture 4 days prior
- Culturing media and conditions (same as test or not): pre-culture medium and test medium are the same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3
Test temperature:
22.4 to 23.1 °C
pH:
Test start: 8.1 to 8.3
Test end: 8.3 to 9.8
Nominal and measured concentrations:
Water accommodated Fractions (WAF) nominal loading rates: Control, 1.0, 3.2, 10 and 32 and 100 mg/L

Measured TOC corrected by the mean value of the control
t=0 hr : n.a, 0.521*, 0.975*, 3.48, 11.0 and 29.3 mg Carbon/L
t=72 hr: n.a, 0.618*, 1.15*, 2.85, 9.56, 28.1 mg Carbon/L

Mean value of all measured samples per treatment group. The results were corrected by the mean value of the control
* TOC content of this loading
Details on test conditions:
TEST CONDITIONS:
- Type and Size: Erlenmeyer flasks of 50 mL volume of test medium containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some mL), kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- Control end cells density: 95.122 [10000/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Light Regime: Continuous illumination
- Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 7555 lux (range: 7100 to 8000 lux)

GROWTH MEDIUM
- Standard medium used: yes - OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
- Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study:Non-GLP pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
65.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 61.3 - 69.7 mg/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
22.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 18.7 - 26.4 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes

- Observation of abnormalities: no -The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal loading rate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to the highest tested loading rate.

Results with reference substance (positive control):
Results with reference substance are valid. Historical ranges not included
72h-ErC50: 1.02 mg/L (95% C.I. 0.984-1.05 mg/L)
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErL50 and the 72 hours EyL50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis (yield) and Weibull analysis (growth rate). For the determination of the 72 hours LOEL and the 72 hours NOEL, the calculated growth rates and yields at each test loading rate were tested for significant differences compared to the control values by Bonferroni-Welch t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical Results:

The TOC content of the test item was determined in the test media samples from all loadings at test start and test end. The highest WAF loading rate 100 mg test item/L was found to contain approximately 28.7 mg carbon/L over the course of the test. The carbon content of the lowest WAF loading rate of 1 mg test item/L was found to be below the Limit of Quantification of 2.5 mg carbon/L. The analytical results show that the WAFs were prepared correctly because of the dose dependent increase of TOC with increasing loading rate. Further the stability of the exposure based on TOC during the test (e.g. no volatilisation) was shown.

Yield y and Percentage Inhibition of y during the Test Period

 

Nominal loading rates [mg test item/L]

Yields y [10000 cells/mL] and % inhibition of y

 

 

0-24 hrs

0-48 hrs

0-72 hrs

 

y

%

y

%

y

%

control

2.196

-

18.93

-

94.622

-

1.0

2.069

5.8

17.025

9.4

89.750

5.1

3.2

1.498

31.8

15.062

19.9

86.663

8.4

10

1.688

23.1*

11.462

39.0*

66.150

30.1*

32

1.308

40.4*

7.600

59.6*

39.119

58.7*

100

0.800

63.6*

1.120

94.0*

1.180

98.8*

 

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* mean value significantly different from the control(tested with Bonferroni-Welch t-test,a = 0.05, one-sided)

 

Growth Rates μ and Percentage Inhibition of μ during the Test Period

Nominal loading rates [mg test item/L]

Growth rates μ [1/day] and % inhibition of μ

 

 

0-24 hrs

0-48 hrs

0-72 hrs

 

μ

%

μ

%

μ

%

control

1.682

-

1.819

-

1.748

-

1.0

1.635

2.8

1.778

2.3

1.732

0.9

3.2

1.373

18.4*

1.718

5.6*

1.720

1.6

10

1.474

12.4*

1.587

12.8*

1.631

6.7*

32

1.285

23.6*

1.392

23.5*

1.457

16.7*

100

0.953

43.3*

0.587

67.7*

0.402

77.0*


negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control
(tested with Williams t-test (24h and 48h) Bonferroni-Welch t-test (72h),a = 0.05, one-sided)

Validity criteria fulfilled:
yes
Remarks:
In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%
Conclusions:
The ErL50, ErL10 and NOELR were 65.4, 22.2 and 3.2 mg test item /L respectively.
Executive summary:

Algae toxicity was assessed in an OECDTG 201 static concentration-response GLP study with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 100, 32, 10, 3.2 and 1.0 mg mg Ginger hot flavor extract (non-volatile) per litre in closed vessels. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. WAFs were considered properly prepared, based on a dose dependent increase of TOC concentrations with loading. Exposure concentrations were considered stable over the test period based on TOC analysis (≥ 82% of initial TOC at 72h, based on quantified loadings). Therefore all reported results refer to nominal loading rates.The ErL50, ErL10 and NOELR for Pseudokirchneriella subcapitata exposed to Ginger hot flavor extract (non-volatile) were 65.4, 22.2 and 3.2 mg test item /L respectively.  

Description of key information

Algae toxicity was assessed in an OECDTG 201 static concentration-response GLP study with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per treatment group were exposed to WAFs prepared at loading rates of 100, 32, 10, 3.2 and 1.0 mg Ginger extract (volatile) per litre in closed vessels.The qualitative analysis of the test item Ginger extract (volatile) in the test samples was performed using gas chromatography with flame ionization detection. From the analytical data it was shown that WAFs were prepared correctly based on increase in peak areas with loading rate. Presence of test item (up to 54% of initial) at test end was also shown for the three highest loading rates. All reported results refer to nominal loading rates.The 72-hour ErL50, ErL10 and NOELR were 40.4, 13.2 and 3.2 mg test item /L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
40.4 mg/L
EC10 or NOEC for freshwater algae:
13.2 mg/L

Additional information

No study information is available regarding the toxicity to aquatic algae of the substance Ginger oil CO2-Total Extract. However, there is sufficient weight of evidence information available from two independent sources to provide appropriate evidence to fulfil the information requirement. Therefore, in line with section 1.2 of Annex XI in regulation (EC) No 1907/2006, a weight of evidence (WoE) approach was used. In this WoE approach the results from OECD TG 201, GLP studies, performed on two qualities of ginger oil extracts (Ginger CO2-SE extract and Ginger oil Hot Flavor CO2-TO extract) were used in order to fulfil the toxicity to aquatic algae endpoint for Ginger oil CO2-Total Extract. These two qualities of ginger oil constitute to the volatile and the non-volatile fraction of the target UVCB (Ginger oil CO2-Total Extract). The two fractions combined cover the constituents present in Ginger oil CO2-Total Extract, albeit in higher concentration ranges in both fractions. By assessing the study results of both fractions in a WoE approach, there is adequate and reliable information available to assess if Ginger oil CO2-Total Extract has or has not a particular dangerous property.

For both extracts, toxicity to aquatic organisms was found in the same order of magnitude (difference less than a factor of 2). For this end-point the selective extract values were selected as worst-case.