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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:

30 January 2018 - 30 November 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: >95% (This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB))
Description: Amber liquid

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples (10 mL) of test media of freshly prepared test media (0 hour) and pooled expired test media (72 hours) were collected in duplicate.
- Sample storage conditions before analysis: Storage stability was not assessed. Samples were extracted and analysed on the day of receipt or sample extracts were stored refrigerated.
- Sample processing: Prior to analysis, the samples were diluted with acetonitrile containing 1% formic acid , then diluting further with diluent (acetonitrile/OECD medium, 1:4, v:v, containing 0.1% formic acid) as required to bring the response within the calibration range.

Vehicle:

no

Details on test solutions:

The dilution media used was OECD medium.

The test material is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test solutions were prepared as Water Accommodated Fractions (WAFs).

The definitive test was conducted at nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The range was based on the results of a range finding test, for which the key results only have been reported. A concurrent control group was also included.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Oban, UK
- Age of inoculum (at test initiation): Concentrated, axenic liquid slope cultures stored under refrigeration (2-8 °C) for up to 8 months.
- Method of cultivation: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 x 10^4 cells/mL.

Test type:

static

Water media type:

other: OECD medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not specified

Test temperature:

23.0 - 23.5 °C

pH:

7.25 - 8.95

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

The definitive test was conducted at nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The range was based on the results of a range finding test, for which the key results only have been reported. A concurrent control group was also included.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile, autoclaved glass 250 mL Erlenmeyer (conical) flasks
- Fill volume: 100 mL
- Aeration: None; shaken continuously (100 rpm) to keep algae in suspension and facilitate transfer
of CO2.
- Renewal rate of test solution: Not applicable; static
- Initial cell density: 1 x 10^4 cells/mL
- Control end cell density: 120 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: Yes; OECD medium

OTHER TEST CONDITIONS
- Intervals of water quality measurement: 0 and 72 hours; temperature also was recorded continuously using minimum/maximum thermometer.
- Sterile test conditions: Yes
- Adjustment of pH: None reported.
- Photoperiod: Constant light
- Light intensity and quality: 4950 – 5850 lux (0 hour); 4900 – 5300 lux (72 hours); under florescent lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: particle counter (Z2 Coulter Counter)
- Chlorophyll measurement: No
- Cell condition: At the end of the test, a subsample of the pooled replicates from the control and each test concentration was taken and the shape and general condition of the algae in each test group was assessed by microscopic observations.

RANGE-FINDING TEST
A range-finding test was conducted at nominal test substance loading rates of 1.0, 10 and 100 mg/L. A concurrent control group was also included. Duplicate test vessels were prepared for the control and each loading rate.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

other: yield

Remarks on result:

other:

Remarks:

(95% confidence limits 1.2 - 2.3)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

(95% confidence limits 56 - 73)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

(95% confidence limits 0.88 - 1.6)

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): None reported
- Test media descriptions: The freshly prepared test solutions solutions at 0 hours appeared as colourless. The appearance of the test media following the 72-hour definitive exposure period were described as non-homogenous hazy dispersion of algae cells with color ranging from very pale green to green, but at 100 mg/L it was a colourless solution.
- Any stimulation of growth found in any treatment: Yes
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

A positive control study was conducted to determine the effects of the reference substance, potassium dichromate, on algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance for 72 hours. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 0.599, 0.604 and 0.875 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate were determined to be 0.32, <0.1 and 0.32 mg/L, respectively.

These results were within the expected toxicity range (growth rate EC50: 0.38 - 2.60 mg/L) for this reference substance and all validity criteria were met; the test is therefore considered valid.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8.

The average specific growth rate (µ) and the final yield over specified time intervals were analysed using Bonferroni Adjusted t Test to determine the no observed effect loading rate (NOEL).

Linear interpolation analysis was performed in order to estimate EL10, EL20 and EL50 values. Where possible, 95% confidence limits were calculated for the EL10, EL20 and EL50 values.

Percent inhibition of growth rate of Pseudokirchneriella subcapitata

Nominal Loading Rate	Average specific growth rate, “µ” (cells x 10^-2/mL)				Inhibition of average specific growth  rates relative to the control (%)
(mg/L)	0-24 hours	24-48 hours	48-72 hours	0-72 hours	0-24 hours	24-48 hours	48-72 hours	0-72 hours
Control	5.81	6.54	7.60	6.65	NA	NA	NA	NA
10	5.39	6.64	6.65	6.23	7	-2	12	6
18	4.47	4.79	3.50	4.26	23	27	54	36
32	4.18	3.70	3.00	3.63	28	43	61	45
56	5.43	4.20	4.44	4.69	7	36	42	29
100	2.66	6.11	-0.281	2.83	54	6	104	57

 NA : Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

 

 

Percent inhibition of yield of Pseudokirchneriella subcapitata

Nominal Loading Rate (mg/L)	Initial cell density (cells x 10^4/mL)	Yield at 72 hours * (cells/mL)	Mean Final Yield per treatment (cells x 10^4/mL)	% Inhibition in Final Yield relative to control
Control	1	1194900 1222300 1256180 1140040 1106860 1217340	119	NA
10	1	768560 803140 1082300	88.5	26
18	1	154780 227300 240600	20.8	83
32	1	105960 128200 147700	12.7	89
56	1	374980 225180 265960	28.9	76
100	1	63320 65700 71120	6.67	94

 *      Final yield (cells/mL) = cell density (cells/mL) at 72 hours – initial cell density (cells/mL)

NA : Not applicable

Validity criteria fulfilled:

yes

Remarks:

The control cell densities increased by a factor of 119. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 15% and the overall CV for specific growth rate was 0.98%.

Conclusions:

Based on nominal loading rates, the 72-hour EyL50 (yield) and the0-72 hour ErL50 (growth rate) values were calculated to be 1.7 and 66 mg/L loading rate WAF, respectively.

The corresponding NOEL for yield and specific growth rate after 72 hours was less than 1.0 mg/L loading rate WAF, the lowest loading rate tested.

Executive summary:

The objective of the study was to determine the effects of the test substance on the growth of the freshwater green alga,Pseudokirchneriella subcapitata,duringa 72-hour growth inhibition toxicity test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

The test material is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test solutions were prepared as Water Accommodated Fractions (WAFs).

The definitive test was conducted at nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The range was based on the results of a range‑finding test, for which the key results were reported. A concurrent control group was also included. Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 x 10^4 algae cells/mL and incubated at 21 - 24°C (under 4440-8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Samples of test media were analysed at 0 (new media) and 72 hours (expired test media). Concentrations of the test substance were determined byhigh performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) usingtheimidazoline-Tconstituent as an analytical marker for quantification. Analysis of the freshly prepared samples at 0 hours resulted in measured concentrations of less than the limit of quantification (LOQ) of the analytical method (assessed as 0.003 mg/L), 0.0505, 0.0898, 0.0623 and 0.163 mg/L for the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs, respectively. The corresponding old pooled media samples resulted in measured concentrations of <LOQ, 0.00796, 0.0151, 0.0110 and 0.0210 mg/L. Given that the test material is a complex UVCB and analytical monitoring was limited to theimidazoline-Tfraction of the test substance, the effect concentrations were based on nominal loading rates.

Based on nominal loading rates, the 72-hour EyL50 (yield) and the0-72 hour ErL50 (growth rate) values were calculated to be 1.7 and 66 mg/L loading rate WAF, respectively. The corresponding NOEL for yield and specific growth rate after 72 hours was less than 1.0 mg/L loading rate WAF, the lowest loading rate tested.

Description of key information

In an OECD 201 study, conducted according to GLP, the effect of the test material on the growth of the freshwater green algae,Pseudokirchneriella subcapitata,was investigated and the 72-hour EL50 value based on growth rate was determined to be 66 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be less than 1.0 mg/L loading rate WAF, the lowest loading rate tested (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

EC50 for freshwater algae:

66 mg/L