Ocimum basilicum, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2017 - 17 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

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)

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.

Storage:
The samples of test start were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed. The samples of test end were analysed immediately after sampling. Afterwards the samples were again stored in a refrigerator (4 ± 4 °C) and will be kept stored up to the date of the final report.

Analyses:
The concentrations of the test item Basil oil were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control

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 concentration 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 117 μL test item into 1050 mL test water, for the test item loading rate of 50 mg test item/L, 59.1 μL test item were mixed into 1060 mL test water, for the loading rate of 25 mg test item/L, 29.5 μg were mixed into 1060 mL test water. The loading rate of 12.5 mg test item/L was prepared by mixing 14.8 μL into 1060 mL test water and for the loading rate of 6.25 mg test item/L, 7.38 μL were mixed into 1060 mL test water. After cessation of mixing and a following period (1 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 concentration. 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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:

21.7 to 22.8 °C

pH:

Test start: 8.1 to 8.3
Test end: 8.4 to 9.7

Nominal and measured concentrations:

Water accommodated Fractions (WAF) nominal loading rates: Control, 6.25, 12.5, 25, 50 and 100 mg/L

Measured TOC corrected by the mean value of the control (n=4)
t=0 hr* : n.a, 3.30, 6.37, 13.02, 25.73 and 51.13 mg Carbon/L
t=72 hr*: n.a, 3.48, 6.61, 12.42, 26.07 and 52.64 mg Carbon/L

*mean value of all measured samples per treatment group per sampling date

Details on test conditions:

TEST CONDITIONS:
- Type and Size: Closed vessel Erlenmeyer flasks of at least 50 mL volume with approximately 50 mL of test medium to minimize the headspace. The vessels were closed with glass stoppers.
- Control end cells density: 68.016 [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 0 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 6402 Lux (range: 5770 to 7100 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: 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.:

23.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval (23.0-23.4 mg test item/L)

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

13.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval (13.6-14.1 mg test item/L)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Value determined by visual inspection of the raw data due to the statistical test appearing to overestimate the biological relevance of the inhibition value

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 this test concentration, the highest concentration tested.

No remarkable observations, clear test medium
pH and temperature were within the limits prescribed in the OECD guideline.

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 a 3-parametric concentration-distribution function. For the determination of the 72 hours LOEL and the 72 hours NOEL, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield and growth rate). The 72 hours LOEL and the 72 hours NOEL for growth rate were finally determined from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical Results:

The quantification of the dissolved fraction of the test item Basil Oil in the test samples was performed by measuring the total organic carbon. During the test the algae were exposed to 51.9, 25.9, 12.7, 6.49 and 3.39 mg Carbon/L (of the nominal loadings of 100, 50, 25, 12.5 and 6.25 mg test item/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	1.188	-	10.414	-	67.516	-
6.25	1.063	10.6	9.171	11.9*	58.922	12.7*
12.5	0.812	31.7*	7.076	32.1*	43.532	35.5*
25	0.686	42.2*	1.251	88.0*	3.759	94.4*
50	0.498	58.0*	0.007	99.9*	0.000	100.0*
100	0.310	73.9*	0.334	96.8*	0.019	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 (72 h) and Williams t-test (24 and 48h), α = 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	1.208	-	1.540	-	1.637	-
6.25	1.138	5.8	1.481	3.9*	1.592	2.7(*)
12.5	0.962	20.3*	1.358	11.8*	1.492	8.9*
25	0.861	28.7*	0.626	59.4*	0.707	56.8*
50	0.687	43.1*	0.007	99.5*	0.000	100.0*
100	0.477	60.5*	0.253	83.6*	0.012	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 (72h) and Williams t-test (24 and 48h), α = 0.05, one-sided), α = 0.05, one-sided)

(*) was significant according to Bonferroni-Welch t-test, but was considered by the study director to be not biologically relevant with an inhibition compared to the control of only 2.7 %.

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 23.2, 13.9 and 6.25 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, 50, 25, 12.5 and 6.25 mg Basil oil 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 analyses (≥ 95% of initial TOC at 72h). Therefore all reported results refer to nominal loading.The 72-hour ErL50, ErL10 and NOELR were determined as  23.2, 13.9 and 6.25 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 group were exposed to WAFs prepared at loading rates of 100, 50, 25, 12.5 and 6.25 mg Basil oil 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 analyses (≥ 95% of initial TOC at 72h). Therefore all reported results refer to nominal loading.The 72-hour ErL50, ErL10 and NOELR were determined as  23.2, 13.9 and 6.25 mg test item /L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

23.2 mg/L

EC10 or NOEC for freshwater algae:

13.9 mg/L

Additional information

Results relate to nominal loading rate, hence are expressed as ErLx.