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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 May 2019 - 26 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
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
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
August 24, 2009
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"
Version / remarks:
February 08, 2019
Qualifier:
according to guideline
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. QA statement)

Test material

Constituent 1
Reference substance name:
Essential oil of Tanacetum annuum
IUPAC Name:
Essential oil of Tanacetum annuum
Test material form:
liquid

Sampling and 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 of all test concentrations and 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, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 72 hours) by pouring together the contents of the test beakers of each treatment. 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 Blue Tansy oil was analysed in the duplicate test media samples from all test concentrations and the control samples from both sampling times (0 and 72 hours).

Test solutions

Vehicle:
no
Details on test solutions:
Dosage of Test Item:
The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on algae, no concentrations above the solubility limit of the test item in test water was tested. Therefore, defined amounts of the test item were added directly to the test water for the five higher test concentration and were carefully stirred for approximately 24 hours in the dark to dissolve as much of the test item as possible.
The highest loading rate of 100 mg test item/L was prepared by mixing 200.8 mg test item into 2008 mL test water, for the loading rate of 32 mg test item/L, 63.7 mg test item were mixed into 1991 mL test water, for the concentration of 10 mg test item/L, 19.9 mg were mixed into 1990 mL test water. The loading rate of 3.2 mg test item/L was prepared by mixing 16.3 mg into 5094 mL test water and for 1.0 mg test item/L, 10.5 mg were mixed into 10500 mL test water. Adequate volumes of the water accommodated fraction of 1.0 mg test item/L were used to prepare the desired loading rates of 0.32 and 0.1 mg/L. After mixing followed a period of 20 minutes of settling to allow phase separation. At the two highest loading rates of 100 and 32 mg/L emulsion drops stayed in the water phase. These loading rates were therefore filtered with a folded filter. The test media of all lower loading rates could be withdrawn from a tap, which was located approx. 2 cm above the bottom after first flushing the tap. These accommodated fractions were used as test media for the test. The test media were prepared just before introduction of the algae (= start of the test).

Control:
In the control, test water was used without addition of the test item.

Appearance of the Test Item in Test Medium:
There were no remarkable observations

Other relevant information:
Phase separation in water was not possible in the loading rates of 100 and 32 mg test item/L. Emulsion drops were still in the water phase. Therefore unsolved test item was separated by a folded filter. There are however no presumed effect on the study. The TOC content of the concentrations was comparable to the pre-test, where the separation from the water phase was possible. The colour of the test medium of the concentrations did not change after filtration.

Test organisms

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
21.8 to 23.0 °C
pH:
Test item treatments at test start: 8.3 to 8.4
Test item treatments at test end: 8.6 to 9.7

Control start: 8.2
Control test end: 9.6
Nominal and measured concentrations:
Water accommodated Fractions (WAF) nominal loading rates: Control, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Measured TOC corrected by the mean value of the control (n=2)
t=0 hr : n.a, t=72 hr: n.a,
Mean value of all measured samples per treatment group. The results were corrected by the mean value of the control (n=2).
Details on test conditions:
TEST CONDITIONS:
- Type and Size: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test medium
- Control end cells density: 77.853 [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: 5093 lux (range: 4570 to 5430 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

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
20.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval 17.0 - 24.9 mg test item/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
3.63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval 2.43 - 5.42 mg test item/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
3.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. interval 2.94 - 3.51 mg test item/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.338 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. interval 0.281 - 0.406 mg test item/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
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 concentration 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 concentration, the highest loading rate tested.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations, clear test medium

- Effect concentrations exceeding solubility of substance in test medium: no


Results with reference substance (positive control):
- Results with reference substance valid? yes * Historical ranges not included
- 72h-ErC50: 0.941 mg/L (95% C.I. 0.910-0.973 mg/L)
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hour ErL50 and the 72 hour EyL50, the corresponding EL20 and EL10 values and where possible their 95 %-confidence limits were calculated by Probit analysis. For the determination of the 72 hour LOEL and the 72 hour NOEL, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Any other information on results incl. tables

Analytical Results:

The quantification of the test item Blue Tansy Oil in the test samples was performed using total organic carbon (TOC) measurement. The TOC content of the test item was determined in the test media samples from all loadings rates at test start and test end. The highest WAF loading rate 100 mg test item/L was found to contain approximately 15 mg carbon/L. The carbon content of the lowest WAF loading rate of 0.1 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) can be concluded from the highest two loading rates.

Yield y and Percentage Inhibition of y during the Test Period

 

Nominal Loading Rate [mg test item/L]

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

0-24 hours

0-48 hours

0-72 hours

y

%

y

%

y

%

Control

2.203

-

16.392

-

77.353

-

0.1

2.163

1.9

15.531

5.3

72.605

6.1

0.32

1.918

13.0

14.044

14.3*

67.566

12.7*

1.0

2.244

-1.9

11.618

29.1*

57.706

25.4*

3.2

1.591

27.8

8.253

49.7*

41.493

46.4*

10

1.020

53.7*

5.123

68.7*

20.898

73.0*

32

0.449

79.6*

1.289

92.1*

3.370

95.6*

100

0.000

100.0*

0.650

96.0*

0.518

99.3*

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 Williams t-test (48 and 72h),a = 0.05, one-sided smaller)

 

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

 

Nominal Loading Rate [mg test item/L]

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

0-24 hours

0-48 hours

0-72 hours

µ

%

µ

%

µ

%

Control

1.686

-

1.758

-

1.682

-

0.1

1.670

1.0

1.733

1.4

1.662

1.2

0.32

1.573

6.7

1.684

4.2

1.637

2.7

1.0

1.677

0.5

1.591

9.5

1.585

5.7

3.2

1.425

15.5*

1.430

18.7*

1.477

12.2*

10

1.109

34.2*

1.208

31.3*

1.251

25.6*

32

0.641

62.0*

0.589

66.5*

0.607

63.9*

100

0.000

100.0*

0.343

80.5*

0.186

89.0*

 

* mean value significantly different from the control(tested with Williams t-test (24, 48 and 72h),a = 0.05, one-sided smaller)

 

Applicant's summary and conclusion

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 20.6, 3.63 and 1.0 mg test item /L respectively.
Executive summary:

Algae toxicity was assessed in an OECDTG 201 static concentration-response test study with closed vessel design. 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 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg Blue Tansy Oil per litre. 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 rate. Exposure concentrations were considered stable over the test period based on TOC analyses concluded from the two highest loading rates. Therefore all reported results refer to nominal values.The 72-hour ErL50, ErL10 and NOELR were determined as  20.6, 3.63 and 1.0 mg test item /L respectively. The 72-hour EyL50 was calculated to be 3.22 mg test item/L, the EyL10 as 0.338 mg test item/L, and the 72-hour NOEyL was determined to be 0.1 mg test item/L.