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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:
28 February 2018 - 02 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: Schinus molle oil
Batch No.: 0008691103
Purity: 100% UVCB
Certificate of Analysis Date: December 19, 2017
Aggregate State at Room Temperature: Liquid
Colour: Colorless to pale yellow
Relative Density D20/4: 0.8475
Expiry Date: 28-Jun-2018
Storage Conditions at Test Facility: At 20 +/- 5 °C, in the dark
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 Schinus molle 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).
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 the algae, no concentrations above the solubility limit of the test item in test water was tested. A defined volume of the test item was added directly to the test water for each test concentration and was carefully stirred for 72 hours in the dark to dissolve as much of the test item as possible. The highest loading rate of 22 mg test item/L was prepared by mixing 27.5 μL test item into 1060 mL test water, for the test item loading rate of 10 mg test item/L, 28.3 μL test item were mixed into 2400 mL test water, for the loading rate of 4.7 mg test item/L, 13.3 μL were mixed into 2400 mL test water. The concentration of 2.2 mg test item/L was prepared by mixing 15.0 μL into 5800 mL test water and for 1.1 mg test item/L, 6.83 μL were mixed into 5800 mL test water. After cessation of mixing and a following period (half 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 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 3 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 22.6 °C
pH:
Test start: 8.2 to 8.3
Test end: 8.4 to 9.6
Nominal and measured concentrations:
Water accommodated Fractions (WAF) nominal loading rates: Control, 1.0, 2.2, 4.7, 10 and 22 mg/L

Measured TOC - corrected by the mean value of the carbon content of test media blank value (TOC blank)
t=0 hr : n.a, n.q*, n.q*, 0.771, 0.846 and 1.69 mg Carbon/L
t=72 hr: n.a, n.q*, n.q*, 0.941, 1.02 and 1.83 mg Carbon/L

* calculation of mean values not considered to be sensible because of negative values. All samples were found to be below the Limit of Quantification. Data is shown for information purposes only.

n.a.: not applicable
n.q.: not quantified
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 of at least 50 mL (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: 91.798 [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: 5642 lux (range: 4840 to 6320 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: 2.2
- 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.:
8.59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 7.45-9.91 mg test item/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
5.64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval: 4.08-7.76 mg test item/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 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 loading rate of 22 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 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 EyL50, the corresponding EyL20 and EyL10 values and where possible their 95 %-confidence limits were calculated by probit analysis using linear weighted regression. The 72 hours ErL50, the corresponding ErL20 and ErL10 values and where possible their 95 %-confidence limits were calculated nonlinear regression using the 3-parametric normal CDF (Cumulative 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 Williams t-test (yield) and Step-down Jonckheere-Terpstra test (growth rate).
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. Due to the limited solubility of the test item the TOC content was found to be below the Limit of Quantification for all samples. Despite these low TOC values, the analytical results provide indications 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 for the highest three loading rates.

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

2.689

-

20.103

-

91.298

-

1.0

2.689

0.0

18.789

6.5

86.576

5.2

2.2

2.431

9.6

15.969

20.6

76.266

16.5*

4.7

2.302

14.4

15.584

22.5*

72.734

20.3*

10

0.755

71.9*

1.483

92.6*

3.718

95.9*

22

0.303

88.7*

0.329

98.4*

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 48h) and Williams t-test (96h), 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

1.847

-

1.855

-

1.737

-

1.0

1.850

-0.2

1.823

1.8

1.719

1.0

2.2

1.767

4.3

1.745

5.9*

1.678

3.4*

4.7

1.721

6.8*

1.733

6.6*

1.661

4.4*

10

0.917

50.3*

0.676

63.5*

0.552

68.2*

22

0.474

74.3*

0.253

86.4*

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 Step-down Jonckheere-Terstra, α = 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 8.59, 5.64 and 1.0 mg test item /L respectively.
Executive summary:

Algal 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 22, 10, 4.7, 2.2 and 1.0 mg Schinus molle 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. Proper WAF preparation and stability of test concentrations during the test (108 -122% of initial TOC at 72h) were indicated. Therefore, all reported results refer to nominal loading rates.The 72-hour ErL50, ErL10 and NOELR were determined as 8.59, 5.64 and 1.0 mg mg test item /L respectively.

Description of key information

Algal 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 22, 10, 4.7, 2.2 and 1.0 mg Schinus molle 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. Proper WAF preparation and stability of test concentrations during the test (108-122% of initial TOC at 72h) were indicated. Therefore, all reported results refer to nominal loading rates.The 72-hour ErL50, ErL10 and NOELR were determined as 8.59, 5.64 and 1.0 mg mg test item /L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
8.59 mg/L
EC10 or NOEC for freshwater algae:
5.64 mg/L

Additional information

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