bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

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:

23 December 2014 to 18 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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:

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.
- Only concentrations within the range to be used for the initial experiments were analysed.

DEFINITIVE TEST - TEST ORGANISM OBSERVATIONS
- Samples were taken at 0, 22, 46 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 test.
- The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

DEFINITIVE TEST - CHEMICAL ANALYSIS OF TEST LOADING RATES
- Samples were taken for quantitative analysis from the control and from the bulk test preparation for each loading rate WAF test group at 0 hours.
- Samples were also taken for quantitative analysis from the pooled replicates at 72 hours.
- All 0-hour samples were stored frozen prior to analysis.
- Duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST
- The loading rate to be used in the initial experiments was determined by a preliminary range-finding test.
- The range-finding test was conducted by exposing Pseudokirchneriella subscapitata cells to nominal loading rates of 0.10 and 1.0 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.
- Due to the need to test at relatively low loading rates, the 0.10 mg/L loading rate WAF was prepared as a serial dilution of the 1.0 mg/L loading rate WAF.
- A nominal amount of test item (20 mg) was added to the surface of 20 L of culture medium to give the 1.0 mg/L loading rate.
- After addition of 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.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one 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 mg/L loading rate WAF.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- A dilution was made from the 1.0 mg/L loading rate WAF to give a 0.10 mg/L loading rate WAF stock solution.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.5 mL) to give the required test concentrations of 0.10 and 1.0 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 Coulter Multisizer Particle Counter.
- The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 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.

INITIAL EXPERIMENTS
- Based on the result of the range-finding test, two initial experiments were conducted at a single loading rate of 1.0 mg/L to confirm that no effect on algal growth occurred.
- In both instances, significant inhibition of growth occurred and it was considered appropriate to conduct the definitive test using a concentration range of 0.10, 0.32 and 1.0 mg/L loading rate WAF.
- A nominal amount of test item (20 mg) was added to the surface of 20 L of culture medium to give the 1.0 mg/L loading rate.
- After addition of 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.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one 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 mg/L loading rate WAF.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further loading rates of 0.32 and 0.10 mg/L.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.1 mL) to give hte required test concentrations of 0.10, 0.32 and 1.0 mg/L loading rate WAF.

Test organisms (species):

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

Details on test organisms:

- The test was carried out using Pseudokirchneriella subscapitata strain CCAP 278/4.
- Liquid cultures of Pseudokirchneriella subscapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, 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 constance 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 10E3 cells/mL.
- The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E4 to 10E5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

24 ± 1 °C

pH:

pH 6.7 to 7.2 during the definitive test (see Table 2, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE-FINDING TEST
- 0.1 and 1.0 mg/L (nominal)

DEFINITIVE TEST
- 0.10, 0.32 and 1.0 mg/L (nominal)

Details on test conditions:

EXPOSURE CONDITIONS
- 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 characterised by a cell density of 6.05 x 10E5 cells/mL. Inoculation of 500 mL of test medium with 4.1 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells/mL and had no significant dilution effect on the final test concentration.
- The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate conducted between 16 September 2014 and 19 September 2014 (see Appendix 2, attached)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Confidence limits 0.29-0.39 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

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

RANGE-FINDING TEST
- Cell densities and percentage inhibition of growth values are given in Table 1 (attached).
- The results showed no effect on groth at 0.10 and 1.0 mg/L loading rate WAF.
- Based on this information, a single loading rate of six replicates of 1.0 mg/L was selected for the initial experiments. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
- Chemical analysis of the 1.0 mg/L loading rate WAF test preparations at 0 and 72 hours (see Appendix 5, attached) showed measured test concentrations of 0.015 and 0.010 mg/L respectively. It was therefore considered possible that the test item was unstable or absorbing to the algal cells present.

INITIAL EXPERIMENTS
- Two initial experiments were donducted at a single nominal loading rate of 1.0 mg/L.
- On both occasions significant inhibition of growth was observed to have occurred suggesting that the effects seen in the range-finding test were erroneous.
- Based on this information, loading rates of 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

CHEMICAL ANALYSIS OF TEST LOADING RATES FOR DEFINITIVE TEST
- Chemical analysis of the test preparations at 0 hours (see Appendix 5) showed measured test concentrations to range from 0.014 to 0.14 mg/L.
- A decline in measured concentration was observed at 72 hours in the range of 0.0076 to 0.043 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.

GROWTH DATA FOR DEFINITIVE TEST
- 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 are given in Table 4 (attached) together with percentage inhibition values.
- The mean cell densities versus time for the definitive test are presented in Figure 1 (attached).
- Percentage inhibition values are plotted against loading rate in Figure 2 and Figure 3 (attached).
- From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72 hour exposure period.

INHIBITION OF GROWTH RATE FOR THE DEFINITIVE TEST
- ErL10 (0-72 h) was determined to be 0.31 mg/L loading rate WAF.
- ErL20 (0-72 h) was determined to be 0.37 mg/L loading rate WAF.
- ErL50 (0-72 h) was determined to be 0.51 mg/L loading rate WAF.
- Where ErLx is the loading rate that reduced growth rate by x %.
- It was not possible to calculate 95 % confidence limits for the ErL50 values as the data generated did not fit the models available for the calculation of confidence limits.

INHIBITION OF YIELD FOR THE DEFINITIVE TEST
- EyL10 (0-72 h) was determined to be 0.17 mg/L loading rate WAF.
- EyL20 (0-72 h) was determined to be 0.22 mg/L loading rate WAF.
- EyL50 (0-72 h) was determined to be 0.34 mg/L loading rate WAF (95 % confidence limits 0.29-0.39 mg/L loading rate WAF).
- Where EyLx is the loading rate that reduced yield by x %.

Reported statistics and error estimates:

INHIBITION OF GROWTH RATE IN THE DEFINITIVE TEST
- Statistical analysis of the growth rate data was carried out for the control 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, 1995).
- There were no statistically significant differences between the control and 0.10 mg/L loading rate WAF (P ≥ 0.05). However, all other loading rates were significantly (P < 0.05) and, therefore, the No Observed Effect Loading Rate (NOEL) based on growth rate was 0.10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.32 mg/L loading rate WAF.

INHIBIBITION OF YIELD IN THE DEFINITIVE TEST
- Statistical analysis of the yield data was carried out as described for assessment of growth rate inhibition.
- There were no statistically significant differences between the control and 0.10 mg/L loading rate WAF (P ≥ 0.05). However, all other loading rates were significantly different (P < 0.05) and, therefore, the No Observed Effect Loading Rate (NOEL) based on yield was 0.10 mg/L loading rate WAF. Correspondingly, the Lowest Observed Effect Loading Rate (LOEL) based on yield was 0.32 mg/L loading rate WAF.

CULTURE MEDIUM

- The culture medium used for the range-finding test, initial experiments and definitive test was the same as that used to maintain the stock culture (see Appendix 3, attached).

PROCEDURE

- Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fractions (WAF).

- A previous study determined the water solubility of the test material to be less than 5.59 x 10E-5 g/L at 20.0± 0.5°C. This suggested that loading rates of greater than 1.0 mg/L were far in excess of that required to ensure the maximum dissolved test item concentration was obtained. Given this, at the request of the sponsor, testing was conducted up to a maximum loading rate of 1.0 mg/L.

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 (see Appendix 4, attached).

CULTURE MEDIUM

- The culture medium used for the range-finding test, initial experiments and definitive test was the same as that used to maintain the stock culture (see Appendix 3, attached).

VALIDATION CRITERIA

- Data show that the cell concentration of the control cultures increased by a factor of 192 after 72 hours (mean cell density of control at 0 h was 4.24 x 10E3 cells/mL; mean cell density of control at 72 h was 8.14 x 10E5 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 12 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean time 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 DURING THE DEFINITIVE TEST

- All test and control cultures were inspected microscopically at 72 hours.

- After 72 hours there were no abnormalities detected in the control or 0.10 mg/L loading rate WAF test cultures.

- Some cell debris and enlarged cells were observed at 0.32 mg/L loading rate WAF.

- No intact cells were observed to be present at 1.0 mg/L loading rate WAF.

WATER QUALITY CRITERIA DURING THE DEFINITIVE TEST

- The pH values of the control and each test concentration are given in Table 2 (attached).

- Temperature was maintained at 24 ± 1 °C throughout the test.

- The pH value of the control cultures (see Table 2, attached) was observed to increase from pH 6.8 to pH 6.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and was therefore within the limits given in the test guideline.

VORTEX DEPTH MEASUREMENTS FOR THE DEFINITIVE TEST

- 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 FOR THE DEFINITIVE TEST

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

- At both the start and end of the mixing period, and following a 1 hour standing period, the 1.0 mg/L loading rate WAF was observed to have formed a clear colourless media column with globules of test item at the surface. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of the test item 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 0.10 mg/L loading rate WAF test cultures were observed to be bright green dispersions.

- The 0.32 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 1.0 mg/L loading rate WAFs were observed to be clear colourless solutions.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave ErL50 (0 -72 h) 0.51 mg/L loading rate WAF. However, it was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for calculation of confidence limits. The No Observed Effect Loading Rate (NOEL) based on growth rate was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.32 mg/L loading rate WAF.
 
Exposure of Pseudokirchneriella subcapitata to the test item gave EyL50 (0 -72 h) 0.34 mg/L loading rate WAF (95 % confidence limits 0.29 -0.39 mg/L loading rate WAF). The No Observed Effect Loading Rate (NOEL) based on yield was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on yield was 0.32 mg/L loading rate WAF.

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 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, a Water Accommodated Fraction (WAF) was prepared.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to Water Accomodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32 and 1.0 mg/L (three replicates 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 test preparations at 0 hours showed that measured test concentrations ranged from 0.014 to 0.14 mg/L. A decline in measured concentration was observed at 72 hours in the range 0.0076 to 0.043 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 Pseudokirchneriella subcapitata to the test item gave ErL50 (0 -72 h) 0.51 mg/L loading rate WAF. However, it was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for calculation of confidence limits. The No Observed Effect Loading Rate (NOEL) based on growth rate was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.32 mg/L loading rate WAF.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave EyL50 (0 -72 h) 0.34 mg/L loading rate WAF (95 % confidence limits 0.29 -0.39 mg/L loading rate WAF). The No Observed Effect Loading Rate (NOEL) based on yield was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on yield was 0.32 mg/L loading rate WAF.

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave ErL50 (0 -72 h) 0.51 mg/L loading rate WAF. However, it was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for calculation of confidence limits. The No Observed Effect Loading Rate (NOEL) based on growth rate was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.32 mg/L loading rate WAF (OECD 201 and EU Method C.3).

Exposure of Pseudokirchneriella subcapitata to the test item gave EyL50 (0 -72 h) 0.34 mg/L loading rate WAF (95 % confidence limits 0.29 -0.39 mg/L loading rate WAF). The No Observed Effect Loading Rate (NOEL) based on yield was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on yield was 0.32 mg/L loading rate WAF (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.51 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 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, a Water Accommodated Fraction (WAF) was prepared.

 

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to Water Accomodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32 and 1.0 mg/L (three replicates 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 test preparations at 0 hours showed that measured test concentrations ranged from 0.014 to 0.14 mg/L. A decline in measured concentration was observed at 72 hours in the range 0.0076 to 0.043 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 Pseudokirchneriella subcapitata to the test item gave ErL50 (0 -72 h) 0.51 mg/L loading rate WAF. However, it was not possible to calculate 95 % confidence limits for the ErL50 value as the data generated did not fit the models available for calculation of confidence limits. The No Observed Effect Loading Rate (NOEL) based on growth rate was 0.10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.32 mg/L loading rate WAF.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave EyL50 (0 -72 h) 0.34 mg/L loading rate WAF (95 % confidence limits 0.29 -0.39 mg/L loading rate WAF). The No Observed Effect Loading Rate (NOEL) based on yield was 0.10 mg/L loading rate WAF and the Lowest Observed EffecLoading Rate (LOEL) based on yield was 0.32 mg/L loading rate WAF.