Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2020 to 19 November 2020

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Range-finding test: A sample of the control and each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen.
- Definitive test: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours. 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 immediate quantitative analysis. A set of duplicate samples was taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

CULTURE MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- The culture medium was prepared using reverse osmosis purified deionized water and the pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Composition of the culture medium is shown in the table below.

Test organisms (species):

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

Details on test organisms:

TEST SYSTEM
- Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10E+03 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 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ± 1 ºC

pH:

- pH values at 0 hours in the definitive test: 7.8 (control); 7.7 (4.5 mg/L); 7.7 (10 mg/L); 7.7 (22 mg/L); 7.7 (45 mg/L) and 7.8 (100 mg/L)
- pH values at 72 hours in the definitive test: 8.4 (control); 8.2 (4.5 mg/L); 8.0 (10 mg/L); 7.8 (22 mg/L); 7.7 (45 mg/L) and 7.6 (100 mg/L)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L test item

DEFINITIVE TEST
- Nominal loading rates of 4.5, 10, 22, 45 and 100 mg/L test item

Details on test conditions:

PURPOSE OF STUDY
- Raphidocelis subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non target organism in freshwater ecosystems.
- 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 2019 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.
VALIDATION OF MIXING PERIOD
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. Details of the investigation were provided in Annex 4 of the full study report.

RANGE-FINDING TEST
- 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 Raphidocelis subcapitata cells to the control and nominal loading rates of 1.0, 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 (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 L of culture medium to give the 1.0, 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 3 hours and the mixtures allowed to stand for 1 hour. A control WAF was prepared under identical conditions but not exposed to the test item. Visual observations made on the test WAFs indicated that a significant amount of dispersed test item was present in the 10 and 100 mg/L water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length) and filter paper. A 10 mL and a 2.0 mL pipette, covered at one end with parafilm, were submerged within the vessels (sealed end down to mid depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the parafilm was removed using the 2.0 ml pipette. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded). Further filtration was conducted using filter paper to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions of test item to be present
- An aliquot (500 mL) of each of the test item loading rate WAFs was separately inoculated with algal suspension (2.4 mL) to give an initial nominal cell density of 5.00 x 10E+03 cells/mL.
- 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 Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ± 1 °C 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 Coulter Multisizer Particle Counter.

DEFINITIVE TEST
- Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 4.5, 10, 22, 45 and 100 mg/L.
- Nominal amounts of test item (22.5, 50, 110, 225 and 500 mg) were each separately added to the surface of 5 L of culture medium to give the 4.5, 10, 22, 45 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 3 hours and the mixtures allowed to stand for 1 hour. A control WAF was prepared under identical conditions but not exposed to the test item. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the 22, 45 and 100 mg/L water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length) and filter paper. A 10 mL and a 2.0 mL pipette, covered at one end with parafilm, were submerged within the vessels (sealed end down to mid depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the parafilm was removed using the 2.0 ml pipette. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration was conducted using filter paper to give the 4.5, 10, 22, 45 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions of test item to be present.
- An aliquot (500 mL) of each of the test item loading rate WAFs was separately inoculated with algal suspension (1.4 mL) to give an initial nominal cell density of 5.00 x 10E+03 cells/mL.
- As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each 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 1.75 x 10E+06 cells per mL. Inoculation of 500 mL of test medium with 1.4 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E+03 cells per mL and had no significant dilution effect on the final test concentration.
- The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ± 1 °C 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.
TEST ORGANISM OBSERVATIONS
- Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10E+03 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.

WATER QUALITY CRITERIA
- The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure using a Hach HQ30d Flexi handheld meter.
- The temperature within the incubator was recorded daily.
- The appearance of the test media was recorded daily.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period.
COMPARISON OF GROWTH RATES
- The average specific growth rate for a specified period was calculated as the logarithmic increase in biomass using the equation µ = ln Nn – ln N1 / tn – t1 where µ = average specific growth rate from time t1 to tn; N1 = cell concentration at t1; Nn = cell concentration at tn; t1 = time of first measurement; tn = time of nth measurement.
- The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
- In addition, the section by section specific growth rate (days 0 to 1, 1 to 2 and 2 to 3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
- Percentage inhibition of growth rate for each replicate test item vessel was calculated using the equation Ir = (µc - µt / µc) * 100 where Ir = percentage inhibition of average specific growth rate; µC = mean average specific growth rate for the control cultures; µt = average specific growth rate for the test culture.

COMPARISON OF YIELD
- Yield was calculated as the increase in biomass over the exposure period using the equation Y = Nn – N0 where Y = yield; N0 = cell concentration at the start of the test; Nn = cell concentration at the end of the test.
- For each test concentration and control the mean value for yield along with the standard deviation was calculated.
- The percentage inhibition of yield was calculated using the equation Iy = [(Yc – Yt) / Yc] * 100 where Iy = percentage inhibition of yield; Yc = mean value for yield in the control group; Yt = mean value for yield for the treatment group.

DETERMINATION OF ELx VALUES
- All ELx values and associated confidence limits and the slope the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood Regression.
- All ELx values were determined using the ToxRat Professional computer software package (TOXRAT).

VALIDATION CRITERIA
- The results of the test are considered valid if the following performance criteria are met:
(i) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
(ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for 72 hour tests) must not exceed 35 %.
(iii) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %.

MAJOR COMPUTERISED SYSTEMS
- The following computerized systems were used in the study:
(i) Cell density determinations: Coulter Multisizer 3 Particle Counter (Version 3.53)
(ii) Statistical analysis: ToxRat (Version 3.3.0)
(iii) Electronic communication system: Veeva QMS

Reference substance (positive control):

yes

Remarks:

potassium dichromate (0.125, 0.25, 0.50, 1.0 and 2.0 mg/L; conducted between 20 January 2020 and 06 March 2020)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits 16 to 31 mg/L loading rate WAF

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limits 7.7 to 12 mg/L loading rate WAF

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: yield

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 3 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 3 hours followed by a 1 hour settlement period.

RANGE-FINDING TEST
- The cell densities and percentage inhibition of growth values from the exposure of Raphidocelis subcapitata to the test item during the range finding test were given in Table 1 of the full study report.
- The results showed no effect on growth rate at 1.0 and 10 mg/L loading rate WAF. However, growth rate was observed to be reduced at 100 mg/L loading rate WAF. Yield was observed to be reduced in all loading rates.
- Based on this information loading rates of 4.5, 10, 22, 45 and 100 mg/L were selected for the definitive test.
- Chemical analysis of the test preparations at 0 hours showed measured boron concentrations (corrected for boron concentrations in the control media) at 10 and 100 mg/L loading rates were 0.12 and 1.1 mg boron/L, corresponding to test item concentrations of 9.7 and 93 mg/L. Chemical analysis of the test preparations at 72 hours showed corrected measured boron concentrations at 10 and 100 mg/L loading rates were 0.14 and 1.1 mg boron/L, corresponding to test item concentrations of 11 and 90 mg/L. Measured concentrations from the 1.0 mg/L loading rate were below the limit of quantification.

DEFINITIVE TEST – CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the test preparations at 0 hours showed measured boron concentrations (corrected for boron concentrations in the control media) were between 0.034 and 1.0 mg boron/L, corresponding to test item concentrations of 2.9 and 87 mg/L.
- Chemical analysis of the test preparations at 72 hours showed corrected measured boron concentrations were between 0.060 and 1.2 mg boron/L, corresponding to test item concentrations of 5.0 and 100 mg/L.

DEFINITIVE TEST – INHIBITION OF GROWTH RATE
- Statistical analysis of the growth rate data was carried out for the control and all loading rates using a Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution.
- There were no statistically significant differences between the control and 4.5 mg/L loading rate WAF (P  0.05); however, all other loading rates were significantly different (P < 0.05) and, therefore the NOEL based on growth rate was 4.5 mg/L loading rate WAF. Correspondingly, the LOEL based on growth rate was 10 mg/L loading rate WAF.

DEFINITIVE TEST – INHIBITION OF YIELD
- Statistical analysis of the yield data was carried out for the control and all loading rates using a Dunnett’s Multiple t-test incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution.
- There were no statistically significant differences between the control and 4.5 mg/L loading rate WAF (P  0.05); however, all other loading rates were significantly different (P < 0.05) and, therefore the NOEL based on yield was 4.5 mg/L loading rate WAF. Correspondingly, the LOEL based on yield was 10 mg/L loading rate WAF.

VALIDATION CRITERIA
- The cell concentration of the control cultures increased by a factor of 110 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.
(i) Nominal cell density of control at 0 hours: 5.00 x 10E+03 cells per mL
(ii) Mean cell density of control at 72 hours: 5.48 x 10E+05 cells per mL
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 13 % 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 to 72 hour) was 2 % 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. After 72 hours there were no abnormalities detected in the control or test cultures at 4.5 mg/L loading rate WAF, however cell debris was observed to be present in the test cultures at 10 mg/L loading rate WAF, some misshapen cells were present in the 22 mg/L loading rate WAF and no intact cells were observed in the 45 and 100 mg/L loading rate WAF. Bacterial contamination was observed in the 22 mg/L loading rate WAF however, inspection of the coulter counts did not support this.

WATER QUALITY CRITERIA
- The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.4 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.

VORTEX DEPTH MEASURMENTS
- 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 WAFs.
- At the start of the mixing period the control was observed to be a clear colorless media column whilst the 4.5, 10, 22, 45 and 100 mg/L preparations were observed to be clear colorless media columns with test item concentrated within the dimple on the surface. After 3 hours stirring and a 1 hour standing period the control was observed to remain as a clear colourless media column, whilst the 4.5 and 10 mg/L preparations were observed to be very slightly hazy media columns with particles of test item dispersed across the surface and one larger lump of test item resting on the surface. The 22 and 45 mg/L loading rates were observed to be slightly hazy media columns with particles of test item dispersed across the surface, with one larger lump of test item resting on the surface in the 22 mg/L loading rate and clusters of larger lumps of test item resting on the surface in the 45 mg/L loading rates. The 100 mg/L loading rate was observed to be a hazy media column with particles of test item dispersed across the surface and an oily slick with large clusters of lumps of test item on the surface. Visual inspection of the WAFs showed a significant amount of dispersed test item to be present in the 22, 45 and 100 mg/L.
- At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72 Hour test period all control and 4.5 mg/L loading rate WAF test cultures were observed to be pale green dispersions, whilst the 10 mg/L loading rate WAF test cultures were observed to be slightly pale green dispersions. All other loading rates were observed to be clear colorless solutions.

Results with reference substance (positive control):

- ErC50 (0 to 72 h): 1.2 mg/L (95 % confidence limits 1.1 to 1.3 mg/L)
- EyC50 (0 to 72 h): 0.51 mg/L (95 % confidence limits 0.45 to 0.58 mg/L)
- No Observed Effect Concentration based on growth rate: 0.25 mg/L
- No Observed Effect Concentration based on yield: 0.125 mg/L
- Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
- Lowest Observed Effect Concentration based on yield: 0.25 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:

- A Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution was carried out on the growth rate data and a Dunnett’s Multiple t-test incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution was carried out on the yield data.
- All statistical analysis was conducted after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the ToxRat Professional computer software package (TOXRAT).

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Raphidocelis subcapitata to the test item gave EL50 values of 22 mg/L loading rate WAF based on growth rate and 9.7 mg/L loading rate WAF based on yield. The corresponding No Observed Effect Loading Rates were determined to be 4.5 mg/L loading rate WAF based on growth rate and yield. The Lowest Effect Loading Rates were 10 mg/L loading rate WAF based on growth rate and yield.

Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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" and 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 study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

 

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of4.5,10,22,45and100 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 boron concentrations (corrected for boron concentrations in the control media) were between 0.034 and 1.0 mg boron/L, corresponding to test item concentrations of 2.9 and 87 mg/L. Chemical analysis of the test preparations at 72 hours showed corrected measured boron concentrations were between 0.060 and 1.2 mg boron/L, corresponding to test item concentrations of 5.0 and 100 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.

CONCLUSION

Exposure of Raphidocelis subcapitata to the test item gave EL50 values of 22 mg/L loading rate WAF based on growth rate and 9.7 mg/L loading rate WAF based on yield. The corresponding No Observed Effect Loading Rates were determined to be 4.5 mg/L loading rate WAF based on growth rate and yield. The Lowest Effect Loading Rates were 10 mg/L loading rate WAF based on growth rate and yield.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The range-finding study was conducted between 11 September 1998 and 14 September 1998 and the definitive study between 2 November 1998 and 6 November 1998.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

acceptable restriction was that analytical measurements on the test material were not conducted.

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

Principles of method if other than guideline:

not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

1998-07-21

Analytical monitoring:

no

Details on sampling:

- Concentrations: Based on the results of the range-finding study the following test concentrations were assigned to the definitive study 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.
- Sampling method: 300 mL of each test sample was extracted with 3 (3 x 50 mL) portions of dichloromethane and the extracts filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue redissolved in methanol (2 mL)
- Stoarage conditions: ambient temperature in light and dark conditions for 72 hours.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study the test material was prepared in the dark as a Water Soluble Fraction (WSF).
Amounts of test material (62.5 and 125 mg) were added to the vortex created in 10 litres of culture medium to give loading rates of 6.25 and 12.5 mg/l. Similarly, amounts of test material (50, 100 and 200 mg) were added to the vortex created in 2 litres of culture medium to give loading rates of 25, 50 and 100 mg/l. Each loading rate was stirred for a period of 23 hours with a vortex depth of approximately 25% of the water column depth.
After 23 hours stirring, the mixtures were allowed to stand for 1 hour prior to the removal of the aqueous phase by siphon and subsequent filtration through 0.45 pm membrane filters to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.
An aliquot (1 litre) of each loading rate WSF was inoculated with algal suspension (5 ml) to give the test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l loading rate WSF.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Age of inoculum (at test initiation): no data
- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21+/- 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15 +) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl. The prepared media was sterilised by 0.22 µm membrane filtration and stored in darkness.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.75 x lo6 cells per ml. This suspension was diluted to a cell density of 2.05 x lo4 cells per ml prior to use. Addition of an aliquot (5 ml) of algal suspension to 1 litre of test medium gave a nominal cell density of 1 x IO4 cells per ml at initiation of the study. The flasks were covered with aluminium foil and incubated (Gallenkamp INR- 401-010W) at 24 f 1 'C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter@M ultisizer II Particle Counter.

- Any deformed or abnormal cells observed:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none.

Hardness:

no data

Test temperature:

24 +/- 1°C

pH:

7.0-8.1

Dissolved oxygen:

no data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentratins: 0, 6.25, 12.5, 25, 50, and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks containing 100 mL of culture medium
- Aeration: yes
- Initial cells density (test): 1.00 x 10^4 cells/mL
- Initial cells density (control): 1.21 x 10^4 cells/mL
- Control end cells density: 2.50 x 10^5 cells/mL
- No. of organisms per vessel: ca. 100 x 10^4 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
NaNO3: 25.5 mg/L
MgCl2.6H2O: 12.164 mg/L
CaCl2.2H2O: 4.41 mg/L
MgSO4.7H2O: 14.7 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.1855 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3.6H2O: 0.159 mg/L
CoCl2.6H2O: 0.00143 mg/L
Na2MoO4.2H2O: 0.00726 mg/L
CuCl2.2H2O: 0.000012 mg/L
Na2EDTA.2H2O: 0.30 mg/L
Na2SeO3.5H2O: 0.000010 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no data
- Other: none.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:0, 1.0, 10.0, 100.0 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 1.0 and
10 mg/l loading rate WSF. However, growth was observed to be reduced at 100 mg/l loading rate WSF.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 13-15

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 15-17

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

none

Results with reference substance (positive control):

not applicable.

Reported statistics and error estimates:

none.

Table 1 and 2 are presented in attached document.

Table 1: Cell densities and pH values un the definitive study.

Table 2: Inhibition of growth rate and biomass.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95 % confidence limits 13 - 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95 % confidence limits 15 - 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF.

Executive summary:

Guideline: A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods: Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to Water Soluble Fraction (WSF) of the test material at loading rates of 6.25, 12.5, 25, 50 and 100 mg/L 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 I I Particle Counter.

Results: Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95% confidence limits 13 - 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95% confidence limits 15 - 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF. The concentration of the components of the test material being taken into the water by the preparation of a WSF were expected to be low given the low water solubility of the test material. Analysis at 0 and 72 hours showed the measured test concentrations to range from 1 % to 34 % of nominal. The variation in measured concentration followed a concentration dependent effect with higher percentage of nominal concentrations being determined from the lower test concentrations. Due to the limited water solubility of the test material in the test media only a small amount of test material can enter the aqueous phase before the test medium becomes saturated. Whilst the amount which can enter the aqueous phase is constant regardless of the amount of test material present it becomes of proportionately greater significance as the nominal loading rate, ie the amount of test material present, decreases. Therefore the measured test concentration, expressed as a percentage of nominal loading rate increased as the loading rate declined.

Description of key information

Key study

Exposure of Raphidocelis subcapitata to the test item gave EL50 values of 22 mg/L loading rate WAF based on growth rate and 9.7 mg/L loading rate WAF based on yield. The corresponding No Observed Effect Loading Rates were determined to be 4.5 mg/L loading rate WAF based on growth rate and yield. The Lowest Effect Loading Rates were 10 mg/L loading rate WAF based on growth rate and yield (OECD 201 and EU Method C.3).

Supporting study

Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95 % confidence limits 13 - 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95 % confidence limits 15 - 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:

22 mg/L

EC10 or NOEC for freshwater algae:

4.5 mg/L

Additional information

Key study

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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" and 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 study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 4.5,10,22,45 and 100 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 boron concentrations (corrected for boron concentrations in the control media) were between 0.034 and 1.0 mg boron/L, corresponding to test item concentrations of 2.9 and 87 mg/L. Chemical analysis of the test preparations at 72 hours showed corrected measured boron concentrations were between 0.060 and 1.2 mg boron/L, corresponding to test item concentrations of 5.0 and 100 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.

CONCLUSION

Exposure of Raphidocelis subcapitata to the test item gave EL50 values of 22 mg/L loading rate WAF based on growth rate and 9.7 mg/L loading rate WAF based on yield. The corresponding No Observed Effect Loading Rates were determined to be 4.5 mg/L loading rate WAF based on growth rate and yield. The Lowest Effect Loading Rates were 10 mg/L loading rate WAF based on growth rate and yield.

Supporting study

Guideline: A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods: Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to Water Soluble Fraction (WSF) of the test material at loading rates of 6.25, 12.5, 25, 50 and 100 mg/L 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 I I Particle Counter.

Results: Exposure of Scenedesmus subspicatus to the test material gave an EbLR50 (72 h) value of 14 mg/L loading rate WSF: 95 % confidence limits 13 to 15 mg/L loading rate WSF and an ErLR50 (0-72 h) value of 16 mg/L loading rate WSF: 95 % confidence limits 15 to 17 mg/L loading rate WSF. The No Observed Effect Loading Rate was found to be 6.25 mg/L loading rate WSF. The concentration of the components of the test material being taken into the water by the preparation of a WSF were expected to be low given the low water solubility of the test material. Analysis at 0 and 72 hours showed the measured test concentrations to range from 1 % to 34 % of nominal. The variation in measured concentration followed a concentration dependent effect with higher percentage of nominal concentrations being determined from the lower test concentrations. Due to the limited water solubility of the test material in the test media only a small amount of test material can enter the aqueous phase before the test medium becomes saturated. Whilst the amount which can enter the aqueous phase is constant regardless of the amount of test material present it becomes of proportionately greater significance as the nominal loading rate, ie the amount of test material present, decreases. Therefore the measured test concentration, expressed as a percentage of nominal loading rate increased as the loading rate declined.