Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

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:

10-Sep-2010 to 10-Oct-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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 the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

reference to same 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

Principles of method if other than guideline:

At the request of the Sponsor, the test consisted of two parts: the test item was tested in part 1 and eventual degradation products of the test item were tested in part 2. Both parts were performed with the single test concentration of nominal 1.0 mg/L, which lies clearly above the water solubility limit of the test item.

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, date of decision 30-apr-2008

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the solvent control and from the control.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
The samples were analyzed immediately after sampling.

The concentrations of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and the potential degradation product 3,3,5-Trimethylcyclo-hexanone were determined in all duplicate test medium, control and solvent control samples.

The analytical procedure and results are described in Appendix I - Analytical Investigations(attached).

Vehicle:

no

Details on test solutions:

The single test concentration of nominal 1.0 mg/L was dosed in two different ways.

Firstly, it was dosed with an organic solvent N,N-dimethylformamide (DMF). The solvent was chosen based on its solubilizing properties and its relative non-toxicity to algae. An application solution was prepared by dissolving 100.44 mg of the test item in 10 mL DMF. For the preparation of the test media, 230 µL of this application solution were added to 2300 mL of test water stirring intensely for 15 minutes. For the preparation of the solvent control, the same volume of DMF was added to the test water (without test item).

In a second approach, a dispersion with the loading rate of 1.0 mg/L was prepared without solvent. The dispersion was prepared by dispersing 2.3779 mg of the test item in 2310 mL of test water and was stirred for 4 hours in order to dissolve a maximum amount of the test item in the test water. During stirring the flask was stoppered with a glass stopper and the air-space was kept at a minimum. The dispersion was left to settle in a separation funnel for about 10 minutes to separate undissolved test material from the test water (as far as possible) and the middle layer was used as test water.
The test media were prepared just before the start of test part 1.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Half of the volume of the test media was immediately used for test part 1. For test part 2, the remaining volumes of test media were left for ageing over a period of 7 days in a closed system to allow the formation of the eventual degradation products. Test part 2 was performed with the 7-day old test media.

The control medium (test water without test item) for test part 2 was also left for ageing over a period of 7 days in a closed system.

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Nygaard et al. recommended describing the taxa within the Genus Raphidocelis HINDAK as:

Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions. The test species are recommended by the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The water temperature during test part 1 was maintained at 23 °C.
The water temperature during test part 2 was maintained at 22 °C.

pH:

At the start of test part 1, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 9.4 to 9.6 were measured (see attached Table 9).
At the start of test part 2, the pH measured in the treatments was 8.0. At the end of the test, pH values of 8.8 to 9.1 were measured (see attached Table 18).

Dissolved oxygen:

not applicable

Salinity:

according to OECD medium

Nominal and measured concentrations:

Please see section "Any other information on materials and methods incl. tables" below.

Details on test conditions:

Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. For furhter information on the test water please see section "Any other information on materials and methods incl. tables".

To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water. The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Glass stoppered 50 mL Erlenmeyer flasks were used (closed system) completely filled with about 60 mL of test medium. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 to 23 °C (test parts 1 and 2) and illuminated by fluorescent tubes (Philips TLD 36W-1/840 for test part 1 and Philips TLD 36W/840 for test part 2), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6480 to 8060 Lux) in the first part of the test and approximately 7000 Lux (range: 6470 to 7500 Lux) in the second part of the test (measured at nine places in the experimental area). The light intensity over the incubation area was within ±15% from the average light intensity as recommended by the guideline.

At request of the Sponsor, the test consisted of two parts: the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide was tested in part 1 and eventual degradation products of the test item were tested in part 2.
Both parts were performed with the single test concentration of nominal 1.0 mg/L, which lies clearly above the water solubility limit of the test item.

Control and solvent control groups were tested in parallel. For test part 2, the control medium (test water without test item) and the solvent control were left (in a closed system) for ageing over a period of 7 days.

The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).

A static test design was applied. The duration of each test part was 72 hours (test parts 1 and 2).

Reference substance (positive control):

yes

Remarks:

twice a year

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 1

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Remarks on result:

other: test part 2

Details on results:

Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on algal growth (test part 1, non-aged test media)
The analytically determined test item concentrations (parent) were as follows:
For further information see section " Any other information on results incl. tables".

At test start, test item concentrations of 0.262 and 0.244 mg/L were measured in both test item treatments of the nominal concentration of 1.0 mg/L (DMF and non-solvent treatment). After the test period of 72 hours, the test item concentrations in the test media had decreased to 0.12 and 0.094 mg/L, respectively (see also analytical results and Table 3 in Appendix I - Analytical Investigations).

However, the analytically determined concentrations of the potential degradation product 3,3,5-trimethylcyclohexanone had not increased significantly over the test period. Concentrations ranged between 0.012 and 0.020 mg/L in both treatments at the start and at the end of the test (see Table 5 in Appendix I - Analytical Investigations).

The reported biological results were based on the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test).

The influence of the test item (dosed with the organic solvent DMF) on the growth of the algae is shown in the attached Table 1 to Table 3 and Figure 1. The influence of the test item on the growth of the algae (dosed without DMF) is shown in the attached Table 5 to Table 7 and Figure 1.

After 72 hours of test duration, the average growth rate was inhibited by 1.0 and 1.2% in the DMF and the non-solvent treatment, respectively. The yield of the algae was inhibited by 4.9 and 5.6%. Inhibitions in the DMF-treatment were not statistically significant, inhibitions in the non-solvent treatment were statistically significant (results of Student-t tests, one-sided, alpha= 0.05, see attached Table 2, Table 3, Table 6 and Table 7). However, inhibitions were at maximum 5.6% and were not estimated as a biologically relevant toxic effect.

Thus, the overall 72-hour NOEC was determined to be the nominal concentration of 1.0 mg/L, corresponding to a mean measured concentration of 0.18 mg/L. The 72 hour LOEC was above the mean measured concentration of 0.18 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item at the tested concentration. Thus, it can be concluded that the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide has no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its water solubility limit under the conditions of the test.

In the solvent control the biomass increased by a factor of 120 over 72 hours (see attached Table 1 and Table 5). The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the solvent control (section-by-section growth rates, see attached Table 4 and Table 8) during 72 hours was 21%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the solvent control after 72 hours was 1.4%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. All validity criteria were also fulfilled for the control.

The algal growth (biomass) in the solvent control was not statistically significantly different from the control after 72 hours (according to a Student-t test, alpha = 0.05, two-sided).
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of test part 1, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 9.4 to 9.6 were measured (see attached Table 9). The water temperature during test part 1 was maintained at 23 °C.


Influence of the test item and eventual degradation products on the algal growth (test part 2, 7-days aged test media):
For further information see section " Any other information on results incl. tables".

At the start of test part 2, test item concentrations of 0.224 and 0.211 mg/L were measured in the 7-days aged test media of the nominal concentration of 1.0 mg/L (DMF and non-solvent treatment, respectively). Thus, the test item concentrations measured at the start of test part 2 were similar to those measured at the start of test part 1 (non-aged test media). After the test period of 72 hours, the test item concentrations in the test media had decreased to 0.052 and 0.051 mg/L, respectively (see also analytical results and attached Table 9 in Appendix I - Analytical Investigations).

However, the analytically determined concentrations of the potential degradation product 3,3,5-trimethylcyclohexanone had not increased significantly over the test period. Concentrations ranged between 0.012 and 0.022 mg/L in both treatments at the start and at the end of test part 2 (see Table 11 in Appendix I - Analyical Investigations) and concentrations were very similar to the 3,3,5-trimethylcyclohexanone concentrations measured in test part 1 (non-aged test media).

Since the measured concentrations of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide as well as the measured concentrations of the potential degradation product 3,3,5-trimethylcyclohexanone differed not significantly between test part 1 and 2, it can be concluded that the test item was fairly stable (in the dark) over the ageing period of 7 days and that the potential degradation product 3,3,5-trimethylcyclohexanone might not have been a main degradation product under the conditions of the test.

Therefore, the biological results of test part 2 were not related to the potential degradation product 3,3,5-trimethylcyclohexanone, but were based on the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of test part 2).

The influence of the test item (dosed with the organic solvent DMF) on the growth of the algae is shown in attached Table 10 to Table 12 and Figure 2. The influence of the test item (dosed without DMF) on the growth of the algae is shown in the attached Table 14 to Table 16 and Figure 2.

After 72 hours of test duration, average growth rate and yield were enhanced by 0.2 and 0.9% in the DMF treatment. In the non-solvent treatment, average growth rate and yield were inhibited by 1.1 and 4.7%. Inhibitions in the DMF-treatment were not statistically significant, inhibitions in the non-solvent treatment were statistically significant (results of Student-t tests, one-sided smaller, alpha= 0.05, see attached Table 11, Table 12, Table 15 and Table 16). However, inhibitions were at maximum 4.7% and were not estimated as a biologically relevant toxic effect.

Thus, the overall 72-hour NOEC was determined to be at the mean measured concentration of 0.11 mg/L (nominal 1.0 mg/L). The 72 hour LOEC was above the mean measured concentration of 0.11 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item at the tested concentrations.Thus, it can be concluded that the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and its eventual degradation products have no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its water solubility limit under the conditions of the test.

In the solvent control the biomass increased by a factor of 81 over 72 hours (see attached Table 1 and Table 14). The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the solvent control (section-by-section growth rates, see attached Table 13 and Table 17) during 72 hours was 18%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the solvent control after 72 hours was 0.7%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. All validity criteria were also fulfilled for the control.

The algal growth (biomass) in the solvent control was not statistically significantly different from the control after 72 hours (according to Student-t test, alpha= 0.05, two-sided).

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of test part 2, the pH measured in the treatments was 8.0. At the end of the test, pH values of 8.8 to 9.1 were measured (see attached Table 18). The water temperature during test part 2 was maintained at 22 °C.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 to 1.7 mg/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values of the test item could not be determined due to the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations.

For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the respective control values by Student-t t-test. Thus, in the non-solvent approach the test item treatment was compared to all control replicates (since the control and the solvent control were not statistically significantly different, “pooled control”). The values of the solvent spiked test item treatment were compared to the solvent control.

Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on algal growth (test part 1, non-aged test media)

The analytically determined test item concentrations (parent) were as follows:

 

Nominal concentration		Measured concentrations at the start of the test (mg/L)		Measured concentrations at the end of the test (mg/L)		Mean measured concentration (mg/L)
1.0 mg/L (DMF)		0.262		0.121		0.18 mg/L
1.0 mg/L (non-solvent)		0.244		0.0936		0.15 mg/L

 

Influence of the test item and eventual degradation products on the algal growth (test part 2, 7-days aged test media)

The analytically determined test item concentrations were as follows:

 

Nominal concentration	Measured concentrations at the start of the test (mg/L)	Measured concentrations at the end of the test (mg/L)	Mean measured concentration (mg/L)
1.0 mg/L (DMF)	0.224	0.0509	0.11 mg/L
1.0 mg/L (non-solvent)	0.211	0.0509	0.10 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Conclusion for test parts 1 and 2:
It can be concluded that the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and its eventual degradation products have no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its limit of solubility in the algae test medium under the conditions of the test.

Executive summary:

The influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 761/2009, C.3.

 

At the request of the Sponsor, the test consisted of two parts: the test item was tested in part 1 and eventual degradation products of the test item were tested in part 2. Both parts were performed with the single test concentration of nominal 1.0 mg/L, which lies clearly above the water solubility limit of the test item.

 

The single test concentration of 1.0 mg/L was dosed in two different ways. Firstly, it was dosed with an organic solvent N,N-dimethylformamide (DMF). In a second approach, a dispersion with the loading rate of 1.0 mg/L was prepared without solvent. This dispersion was stirred for 4 hours in order to dissolve a maximum amount of the test item in the test water. After the stirring period possibly undissolved test item was removed. Therefore, the dispersion was left to settle for about 10 minutes and the middle layer was used as test medium.

 

The so prepared test media were used immediately for test part 1. For test part 2, the test media were left (in a closed system) for ageing over a period of 7 days to allow the formation of the eventual degradation products. Test part 2 was performed with the 7-day old test media.

 

Control and solvent control groups were run in parallel. For test part 2, the control medium (test water without test item) and the solvent control were left (in a closed system) for ageing over a period of 7 days.

 

To avoid any losses of test item, the test was performed in tightly closed test flasks (closed system) completely filled with test medium to minimize evaporation of the test item.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

Results of test part 1:

At test start, test item concentrations of 0.262 and 0.244 mg/L were measured in both test item treatments of the nominal concentration of 1.0 mg/L (DMF and non-solvent treatment).After the test period of 72 hours, the test item concentrations in the test media had decreased to 0.12 and 0.094 mg/L, respectively (see also analytical results and Table 3 in attached Appendix I - Analytical Investigations).

 

The reported biological results were based onthe mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end oftest part 1):

 

Nominal concentration	Measured concentrations at the start of the test (mg/L)	Measured concentrations at the end of the test (mg/L)	Mean measured concentration (mg/L)
1.0 mg/L (DMF)	0.262	0.121	0.18 mg/L
1.0 mg/L (non-solvent)	0.244	0.0936	0.15 mg/L

 

The analytically determined concentrations of the potential degradation product 3,3,5-trimethylcyclohexanone had not increased significantly over the test period. Concentrations ranged between 0.012 and 0.020 mg/L in both treatments at the start and at the end of test part 1.

 

The inhibition of the algal growth (average growth rate and yield) was at maximum 5.6% in all test item treatments of the single test concentration of nominal 1.0 mg/L.

 

Therefore, the overall 72-hour NOEC of test part 1 was determined to be at the mean measured concentration of 0.18 mg/L (nominal 1.0 mg/L).

 

 

Results of test part 2:

At the start of test part 2, test item concentrations of 0.224 and 0.211 mg/L were measured in the 7-days aged test media of the nominal concentration of 1.0 mg/L (DMF and non-solvent treatment, respectively). Thus, the test item concentrations measured at the start of test part 2 were similar to those measured at the start of test part 1 (non-aged test media).After the test period of 72 hours, the test item concentrations in the test media had decreased to 0.052 and 0.051 mg/L, respectively.

 

The reported biological results were based onthe mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end oftest part 2):

 

Nominal concentration	Measured concentrations at the start of the test (mg/L)	Measured concentrations at the end of the test (mg/L)	Mean measured concentration (mg/L)
1.0 mg/L (DMF)	0.224	0.0516	0.11 mg/L
1.0 mg/L (non-solvent)	0.211	0.0509	0.10 mg/L

 

 

The analytically determined concentrations of the potential degradation product 3,3,5-trimethylcyclohexanone had not increased significantly over the test period. Concentrations ranged between 0.012 and 0.022 mg/L in both treatments at the start and at the end of test part 2 and concentrations were very similar to the 3,3,5-trimethylcyclohexanone concentrations measured in test part 1 (non-aged test media).

 

The inhibition of the algal growth (average growth rate and yield) was at maximum 4.7% in all test item treatments of the single test concentration of nominal 1.0 mg/L. Therefore, the overall 72-hour NOEC of test part 2 was determined to be at the mean measured concentration of 0.11 mg/L (nominal 1.0 mg/L).

Conclusion for test parts 1 and 2: It can be concluded that the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and its eventual degradation products have no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its limit of solubility in the algae test medium under the conditions of the test.

Description of key information

Based on the available study results, the inhibition of the algal growth (average growth rate and yield) was at maximum 4.7%. Therefore, the overall

72-hour NOEC of test part 2 was determined to be at the mean measured concentration of 0.11 mg/L (nominal 1.0 mg/L), the highest concentration possible to test under the conditions of the assay. It was not possible to determine an EC50 at this concentration that is itself significantly higher than the water solubility limit. Therefore, these results indicate that the substance is unlikely to be toxic to algae and cyanobacteriae at its limit of solubility.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.11 mg/L

Additional information

One valid study on algae was well conducted and presented strong data (Weber 2010). This experiment was performed as follows:

OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 761/2009, C.3. The inhibition of the algal growth (average growth rate and yield) was a maximum of 4.7% in all test item treatments of the single test concentration of nominal 1.0 mg/L. Therefore, the overall 72-hour NOEC of test part 2 was determined to be at the mean measured concentration of 0.11 mg/L (nominal 1.0 mg/L), the highest concentration possible to test under the conditions of the assay and considerably higher than the water solubility.

Therefore, these results indicate that the substance is not potentially toxic to algae and cyanobacteriae under the conditions of the study.