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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance is a mixture that is poorly soluble in water (information from sponsor). Therefore a water accommodated fraction (WAF) was prepared using a slow-stir liquid-liquid saturator technique. Two glass aspirator bottle (2-L) with a bottom side-outlet attached to a stopcock were filled with 2-L test media. The necessary volume of the test substance was calculated from the density. Then 267-µL (100 mg/L considering density = 0.75 g/cm³) per bottle of the test substance was pipetted carefully on the water surface. The bottles were closed and the solutions were stirred on a magnetic plate for approximately 1 day. The stirring was slow (approx. 130 rpm) so that no vortex formed. The undissolved test substance remained on the surface and the required volume of the WAF (approx. 1.5-L) was removed from the bottom of the bottles and again visually inspected for the presence of any undissolved test substance. The saturated solutions from both bottles were combined and used for testing.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved test substance was observed in the merged test solution. The test solution was visibly clear following preparation.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): collection of algal cultures in Göttingen, Germany
- Method of cultivation: A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 °C
pH:
7.4 -7.5 (at test start)
7.4 - 10.0 (at test end)
Nominal and measured concentrations:
Nominal: 100 mg/L as loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) containing 100 mL test solution plugged with gas permeable silicone sponge caps
- Type (delete if not applicable): closed
- Initial cells density: 0.3E4 cells/mL
- Control end cells density: 321E4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- other: continuous stirring

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Alkalinity: To accommodate testing in a closed system (see Mayer 2000; ISO/DIS 14442, 2004) the concentration of NaHCO3 in the final modified OECD medium was increased to 300 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH at start and at end of test, temperature continuously

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: permanent illumination
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25). To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily. Average 5666 lux (within ± 15% variability) at a wave length of 400 - 700 nm. Light intensity was adjusted to the lower end of the acceptable range, to control pH changes due to excessive algal growth in the closed test vessels.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
- Chlorophyll measurement: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm).
- other: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

TEST CONCENTRATIONS
- Range finding study: yes
- Results used to determine the conditions for the definitive study: In the preliminary range finding test a concentration of 100 mg/L caused no inhibition of algal growth relative to the control after 72 hours. According to the OECD-guideline, the highest suggested test concentration is 100 mg/L for a limit test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: the treatment show a slightly higher growth rate (+4.8%) compared to the control.
Results with reference substance (positive control):
The ErC50 (72 h) of the control substance potassium dichromate was 1.05 mg/L (Date of the last control experiment: 13 Feb 2015 , project number: 60E0063/04E006). These results indicate that the algae are responding normally to toxicant stress.
An additional reference toxicant test was conducted with the same method modifications described in this study (modified exposure media, closed test system, and reduced inoculation density) to determine any effect on algal response to toxic stress. The ErC50 (72 h) of the control substance potassium dichromate was 1.32 mg/L (Date of experiment: 23 Feb 2015 , project number: (60E0063/04E007). These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study.
Validity criteria fulfilled:
yes
Conclusions:
ErL50 (72 h) > 100 mg/L
NOELR (72 h) ≥ 100 mg/L
Executive summary:

ErL50 (72 h) > 100 mg/L

NOELR (72 h) ≥ 100 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
31 July 2014 to 13 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of rleevant results.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Test Organism Observations
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

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.
Vehicle:
no
Details on test solutions:
 Experimental Preparation
A nominal amount of test item (230 mg) was added to the surface of 2.3 liters of culture medium in a sealed vessel with minimal headspace to give the 100 mg/L loading rate. 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 mixture 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 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
 
An aliquot (2 liters) of the 100 mg/L loading rate WAF was inoculated with algal suspension (17 mL) to give the required test concentration of 100 mg/L loading rate WAF.
 
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.

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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 C until the algal cell density was approximately 104 – 105 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not stated in report
Test temperature:
24 +/- 1°C recorded daily
pH:
Measured using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen:
Not stated in report
Salinity:
Not applicable as a freshwater study
Nominal and measured concentrations:
Range finding test: 10 and 100 mg/l loading rate
Definitive test: 100 mg/l loading rate
Details on test conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and 100 mg/L loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.85 x 105 cells per mL. Inoculation of 2 liters of test medium with 17 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Details on results:
From the data given in Tables 2 and 4 (please refer to the attached documents), it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.
Results with reference substance (positive control):
A positive control (Harlan Study Number 41303826) 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 – 72 h) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) 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.

Reported statistics and error estimates:
Inhibition of growth rate
ErL10 (0 - 72 h) : >100 mg/L loading rate WAF
ErL20 (0 - 72 h) : >100 mg/L loading rate WAF
ErL50 (0 - 72 h) : >100 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases in growth rate (P0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.


Inhibition of Yield
EyL10 (0 - 72 h) : >100 mg/L loading rate WAF
EyL20 (0 - 72 h) : >100 mg/L loading rate WAF
EyL50 (0 - 72 h) : >100 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as per the above. There were no statistically significant decreases in yield between the control and 100 mg/L loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 97 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

 

Mean cell density of control at 0 hours         :          5.39 x 103cells per mL

Mean cell density of control at 72 hours       :          5.22 x 105cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 24% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

 

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

 

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

 

The pH value of the control cultures (see Table 2) was observed to increase from pH 8.6 at 0 hours to pH 10.5 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth (see Figure 1) exceeding the transfer rate of CO2from the gaseous phase to the aqueous phase. In this situation CO2required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

 

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

  

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF.

 

At both the start and end of the mixing period, and following a 1-Hour standing period, the WAF was observed to have formed a clear colorless media column with an oily globule of test item floating at the media surface. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

 

At the start of the test all control and 100 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. After the 72 -Hour test period all control and test cultures were observed to be green dispersions.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction

A 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 a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Hermanet al. 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

 

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…….

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured concentration of 3.96 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 0.98 mg C/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 EL50values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

There is no data available for this substance. However, key and supporting data is available for structural analogues Hydrocarbons, C11-C13 (odd number), n-alkanes, <2% aromatics,

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2%, Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics and Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2. The data is read across to this substance based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13. Key and supporting information is summarised below:

Hydrocarbons, C11-C13 (odd number), n-alkanes, <2% aromatics presented a 72-h EL50 (growth rate) for Pseudokirchneriella subcapitata of >100 mg/L (WAF) and a 72-h NOELR (growth rate) for Pseudokirchneriella subcapitata of 100 mg/L (WAF).

 

Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics presented a 72-h EL50 (growth rate) for Pseudokirchneriella subcapitata of >100 mg/L (WAF) and a 72-h NOELR (growth rate) for Pseudokirchneriella subcapitata of 100 mg/L (WAF).

 

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate and biomass) for Skeletonema costatum of >100,000 mg/L (WAF) and a 72-h NOELR (growth rate and biomass) for Skeletonema costatum of 100,000 mg/L (WAF).

 

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate) for Skeletonema costatum of >100,137 mg/L (WAF) and a 72-h EL10 (growth rate) for Skeletonema costatum from 60021 to 80072 mg/L (WAF).

 

Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate and biomass) for Pseudokirchneriella subcapitata of >1,000 mg/L (WAF) and a 72-h NOELR (growth rate and biomass) for Pseudokirchneriella subcapitata of 1,000 mg/L (WAF).

 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate and biomass) for Pseudokirchneriella subcapitata of >1,000 mg/L (WAF) and a 72-h NOELR (growth rate and biomass) for Pseudokirchneriella subcapitata of 1,000 mg/L (WAF).

 

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 72-h EL50 (growth rate and biomass) for Pseudokirchneriella subcapitata of >1,000 mg/L (WAF) and a 72-h NOELR (growth rate and biomass) for Pseudokirchneriella subcapitata of 1,000 mg/L (WAF).

Key value for chemical safety assessment

Additional information

Measured toxicity data are available for Hydrocarbons, C11-C13 (odd number), n-alkanes, <2% aromatics;  Hydrocarbons, C12-C15, n-alkanes, isoalkanes, <2% aromatics; Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics; and Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics.

Six studies followed an OECD Guideline 201 (Alga, Growth Inhibition Test) using Pseudokirchneriella subcapitata as test organism, and two studies followed an ISO 10253 (Water quality - Marine Algal Growth Inhibition Test) using Skeletonema costatum as test organism. No test showed a statistically significant effect on growth rate or biomass at the highest tested concentrations, which ranged from 100 to >100,000 mg/L (WAF). Therefore, the substances presented a 72-h EL50 (growth rate and/or biomass) of >100 mg/L (WAF) and a 72-h NOELR (growth rate and/or biomass) of ≥ 100 mg/L (WAF).