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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:

2018-02-01 - 2018-03-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Method C.3 of Commission Regulation (EC) 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15 to 25 ºC) without protection from light

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Due to the need to test at concentrations below 1.0 mg/L where accuracy in the weighing of the test item may be compromised, the loading rates of 0.10 and 0.32 mg/L were prepared as serial dilutions from the 1.0 and 3.2 mg/L loading rates WAFs respectively. First, the test item is a viscous liquid, which may not be completely expelled from from the end of the disposable syringe it was weighed in due to viscosity, so it cannot be guaranteed that the nominal concentrations were reached. Second, the maximum volume of media which can be prepared is technically limited, so the weighing of higher test item amounts and subsequent preparation of the WAFs is limited, too.
Nominal amounts of test item (10, 32 and 100 mg) were each separately added to the surface of 10 liters of culture medium to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. Dilutions were made from the 1.0 and 3.2 mg/L loading rate WAFs to give further test concentrations of 0.10 and 0.32 mg/L loading rate WAF respectively.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.9 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata strain CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10E4 to 10E5 cells/mL.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

24 ±1 ºC

pH:

The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Nominal: 0, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Measured: See below, results section & attachment

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 ml fill volume
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.49 x 10E5 cells per mL. Inoculation of 500 mL of test medium with 3.9 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.
- Control end cells density: 1.67E+06 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux, provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 20, 43 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10E3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 2.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Nominal amounts of test item (20, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 2.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 2.0, 10, 100 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.3 mL) to give the required test concentrations of 2.0, 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Due to a technical oversight a 2.0 mg/L loading rate WAF, rather than a 1.0 mg/L loading rate WAF was prepared in error. This was considered to have had no adverse effect on the outcome of the test.

Reference substance (positive control):

yes

Remarks:

A separate positive control study used potassium dichromate as the reference item.

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits (mg/L Loading Rate WAF) = 3.0 - 4.9

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

NOEL

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

LOEL

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95% Confidence Limits (mg/L Loading Rate WAF) = 0.64 - 0.98

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

NOEL

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

biomass

Remarks:

yield

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

LOEL

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

biomass

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Colour differences: After the 72-Hour test period all control, 0.10 and 0.32 mg/L loading rate WAF test cultures were observed to be green dispersions. The 1.0 mg/L test cultures were light green dispersions, the 3.2 mg/L test cultures were very pale green dispersions whilst the 10 mg/L test cultures remained as clear colorless solutions. Green coloration of the test cultures occurred due to algal growth.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Substance was applied as WAF

Results with reference substance (positive control):

A separate positive control study used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the substance towards aquatic algae. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.12 to 8.4 mg/L. Analysis of the test preparations at 72 hours showed measured test concentrations to range from 0.11 to 8.5 mg/L.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
The ErL50(72h) was determined to be 3.9 mg/l. According to Regulation (EC) No.1272/2008 and amendments (CLP), the criteria for classification of a substance in category Acute 1 are defined on the basis of acute aquatic toxicity data only (EC50, EL50, LC50 or LL50); classification is required for values ≤ 1 mg/L. Therefore, the substance does not need to be classified for acute (short-term) aquatic hazard. However according to Table 4.1.0 (Commission Regulation (EU) No 286/2011 of 10 March 2011), Classification categories for hazardous to the aquatic environment, (iii) Substances for which adequate chronic toxicity data are not available, and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log K ow ≥ 4), classification as Category Chronic 2 is required if the following short-term results are obtained:
96 hr LC 50 (for fish) > 1 to ≤10 mg/l and/or
48 hr EC 50 (for crustacea) > 1 to ≤10 mg/l and/or
72 or 96 hr ErC 50 (for algae or other aquatic plants) > 1 to ≤10 mg/l
With an ErL50(72h) of 3.9 mg/ml, and the fact that no biodegradation observed, 0% (O2 consumption) in 28 days (OECD TG 301D), the substance must be classified as Aquat. Chron. Cat. 2.

Executive summary:

Introduction

A GLP study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range‑finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of0.10,0.32,1.0,3.2and10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Results

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.12 to 8.4 mg/L. Analysis of the test preparations at 72 hours showed measured test concentrations to range from 0.11 to 8.5 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	3.9	3.0	-	4.9	0.32	1.0
Yield	0.79	0.64	-	0.98	0.10	0.32

 

The substance must be classified as Aquat. Chron. Cat. 2.

Description of key information

Acute Toxicity to Aquatic Algae: ErL50(72h) = 3.9 mg/l, NOELr(72h) = 0.32 mg/l, LOELr(72h) = 1.0 mg/l for Pseudokirchneriella subcapitata (WAF, OECD 201, EU method C.3, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

3.9 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L