Rape oil, reaction products with diethylenetriamine

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:

25 April 2016 to 26 May 2016

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

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:

RANGE FINDING TEST
- 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.

DEFINITIVE TEST
- Samples were taken from bulk test preparation at 0 hours for the control and each loading rate WAF test group.
- Samples were also taken from 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:

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 is defined in Annex 3 (attached).

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 Water Accommodated Fraction (WAF) of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work (see Annex 4, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

Test organisms (species):

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

Details on test organisms:

TEST SYSTEM
- The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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 ± 2 °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 10 x E03 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 10E04 to 10E05 cells/mL.
- A positive control used potassium dichromate as the reference item. Details of the positive control are given in Annex 2 (attached). The positive control was conducted between 07 December 2015 and 10 December 2015.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ± 1 °C

pH:

7.2 to 7.9 in the definitive test

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 10 and 100 mg/L

DEFINITIVE TEST
- Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

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 t0; 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 0-1, 1-2 and 2-3) 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
- ELx values were determined by inspection of the growth rate and yield data after 72 hours.

STATISTICAL ANALYSIS
- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

VALIDATION CRITERIA
- The results of the test are considered valid if the following performance criteria are met:
(i) 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 0-1, 1-2 and 2-3, 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%.

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Annex 4, attached) indicated that there was no increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- For the purpose of testing the WAF was therefore prepared using a stirring period of 23 hours followed by a 1-hour settlement period.

RANGE FINDING TEST
- The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 (attached).
- Results showed no effect on growth rate at 10 and 100 mg/L loading rate WAF. However, yield appeared to be slightly reduced at both 10 and 100 mg/L loading rate WAF.
- Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
- Chemical analysis of the test preparations at 0 and 72 hours (see Annex 5, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0087 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

CHEMICAL ANALYSIS OF DEFINITIVE TEST LOADING RATES
- Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours (see Annex 5, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0087 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
- 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.

DEFINITVE TEST GROWTH DATA
- Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (attached).
- Daily specific growth rates for the control cultures are given in Table 3 (attached).
- Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (attached).
- Mean cell densities versus time for the definitive test are presented in Figure 1 (attached).
- From the data given in Tables 2 and 4 (attached), 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.

VALIDATION CRITERIA
- The cell concentration of the control cultures increased by a factor of 166 after 72 hours (mean cell density of control at 0 hours was 5.00 x 10E03 cells/mL and mean cell density of control at 72 hours was 8.30 x 10E05 cells/mL).
- 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.
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 6 % 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 1 % 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 at 72 hours.

WATER QUALITY CRITERIA
- The pH values of the control and each test concentration are given in Table 2 (attached).
- Temperature was maintained at 24 ± 1 ºC throughout the test.

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 WAFs.
- At the start of the mixing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear colourless water columns with one waxy string of test item floating on the surface. After 23 hours stirring and a 1-hour standing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to remain as at the start of stirring. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

- Results from the positive control with potassium dichromate were within the normal ranges for this reference item (see Annex 2, attached).

Reported statistics and error estimates:

INHIBITION OF GROWTH RATE
- The ErL10 (0-72h), ErL20 (0-72h) and ErL50 (0-72h) values were all reported as > 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 and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).
- There were no statistically significant differences (P ≥ 0.05), between the control and each loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was considered to be 100 mg/L mg/L loading rate WAF.

INHIBITION OF YIELD
- The EyL10 (0-72h), EyL20 (0-72h) and EyL50 (0-72h) values were all reported as > 100 mg/L loading rate WAF where EyLx is the loading rate that reduced yield rate by x %.
- Statistical analysis of the yield data was carried out as described for growth rate.
- There were no statistically significant differences between the control and each loading rate WAF test group (P ≥ 0.05). Therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values > 100 mg/L loading rate WAF based on growth rate and yield. The No Observed Effect Loading Rate (NOEL) was therefore 100 mg/L 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 algaPseudokirchneriella 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 subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 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

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method were obtained, which was determined to be 0.0087 mg/L. This does not infer that there was no test item in solution, only that which was, was present at a concentration of less than the LOQ. Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

 

CONCLUSION

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values > 100 mg/L loading rate WAF based on growth rate and yield. The No Observed Effect Loading Rate (NOEL) was therefore 100 mg/L based on growth rate and yield.

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values > 100 mg/L loading rate WAF based on growth rate and yield. The No Observed Effect Loading Rate (NOEL) was therefore 100 mg/L based on growth rate and yield.

(OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella 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 subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 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

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method were obtained, which was determined to be 0.0087 mg/L. This does not infer that there was no test item in solution, only that which was, was present at a concentration of less than the LOQ. Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

 

CONCLUSION

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values > 100 mg/L loading rate WAF based on growth rate and yield. The No Observed Effect Loading Rate (NOEL) was therefore 100 mg/L based on growth rate and yield.