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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:
04 October 2017 - 16 January 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)
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 Geranium oil were 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
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 72 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 118.31 μL test item into 1050 mL test water, for the test item loading rate of 31.6 mg test item/L, 37.39 μL test item were mixed into 1050 mL test water, for the loading rate of 10 mg test item/L, 11.83 μg were mixed into 1050 mL test water. The loading rate of 3.2 mg test item/L was prepared by mixing 3.79 μL into 1050 mL test water and for the loading rate of 1.0 mg test item/L, 1.18 μL were mixed into 1050 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 over a period of 72 hours, before introducing 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:
21.9 to 23.5 °C
pH:
Test start: 7.7 to 8.2
Test end: 8.0 to 9.6
Nominal and measured concentrations:
Water accommodated Fractions (WAF) nominal loading rates: Control, 1.0, 3.2, 10, 31.6 and 100 mg/L

Measured TOC corrected by the mean value of the control (n=4)
t=0 hr* : n.a, -0.17, 1.19, 5.36, 14.83 and 49.67 mg Carbon/L
t=72 hr*: n.a, 0.49, 2.28, 6.02, 14.41 and 48.41 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: 86.237 [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: 5343 Lux (range: 4920 to 5770 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) :
- Determination of cell density: 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.:
30.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval ( 29.0-31.8 mg test item/L)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
15.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. interval ( 12.9-19.2 mg test item/L)
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 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

- Algicidal Effects:
The test item causes not only inhibitory or algistatic but also algicidal effects as the cell density decreased to a lower level than at test start. This was observed at the highest test levels of 31.6 and 100 mg/L.

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 level of 10.0 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 two highest loading rates of 31.6 and 100 mg test item/L caused strong inhibitory effects on the algal. In these replicates no algae cells could have been observed.

Clear test medium and 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 however 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 using a three parametric normal 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 using Bonferroni-Welch t-test (yield) and Bonferroni-Holm t-test (growth rate). 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 Geranium oil in the test samples was performed by measuring the total organic carbon content. During the test the algae were exposed to 49.0, 14.6, 5.69 and 1.74 mg Carbon /L (of the nominal loadings of 100, 31.6, 10 and 3.2 mg test item/L). The results of the control and the lowest nominal concentration were below LOQ (1 mg TOC/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

1.840

-

15.718

-

85.737

-

1.0

2.036

-10.7

17.283

-10.0

88.927

-3.7

3.2

2.036

-10.7

16.484

-4.9

89.682

-4.6

10

1.185

35.6

14.352

8.7

76.921

10.3

31.6

0.400

78.3*

1.894

88.0*

5.433

93.7*

100

0.000

100*

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), 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

1.543

-

1.738

-

1.717

-

1.0

1.615

-4.7

1.785

-2.7

1.728

-0.7

3.2

1.623

-5.2

1.755

-0.9

1.729

-0.7

10

1.166

24.4

1.693

2.6

1.680

2.1

31.6

0.588

61.9*

0.783

55.0*

0.811

52.8*

100

0.000

100.0*

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 multiple sequential t- test (48h), 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 30.4, 15.7 and 10 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 1.0, 3.2, 10, 31.6 and 100 mg Geranium 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 (≥ 97% of initial TOC at 72h). Therefore all reported results refer to nominal values.The 72-hour ErL50, ErL10 and NOELR were determined as 30.4, 15.7 and 10 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 1.0, 3.2, 10, 31.6 and 100 mg Geranium 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 (≥ 97% of initial TOC at 72h). Therefore all reported results refer to nominal values.The 72-hour ErL50, ErL10 and NOELR were determined as 30.4, 15.7 and 10 mg test item /L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
30.4 mg/L
EC10 or NOEC for freshwater algae:
15.7 mg/L

Additional information

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