1,6-Bis(methoxybenzoyloxy)hexane

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:

Start: 12 January 2004 Completion: 12 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study procedures were based on the ISO International Standard 8692: 'Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricomutum", First edition, 15 November 1989. In addition, the procedures were designed to meet the test methods and validity criteria prescribed by the following guiidelines: - European Economic Community (EEC), EEC Directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, C-3: "Algal Inhibition Test" adopted December 1992. - Organization for EconomiG Co-operation and Development (OECD), OECD guideline for Testing of Chemicals, guicieline No. 201: "Algae, Growth Inhibition Test", Adopted June 7, 1984.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Remarks:

GLP Statement date (s): 30 March 2004

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Test samples - limit test
The concentrations at the start of the test were 99%-104 % of nominal. The concentrations decreased by more than 20% during the test. At the end of the test, the concentrations were 3% and 9% of initial.

- Sampling method:
During the limit test duplicate samples were taken from the test concentration and the blankcontrol.
Frequency: at t=O h, t=24 hand t=72 h
Volume: 10ml
Storage: Samples were transferred to Analytical Chemistry for analysis directly after sampling

- Sample storage conditions before analysis:
Each sample (10 ml) was supplied in a 20 ml volumetric flask. The flasks were filled up to the mark with acetonitrile.

Vehicle:

yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture.
- Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae culture
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ±2°C.

- Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

- Stock culture medium: M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/I
K2HPO4 40 mg/I
MgS04.7H2O 76 mg/I
Na2CO3 10H2O 54 mg/I
C6H8O7.H2O 6 mg/I
NH4NO3 330 mg/I
CaCl2.H2O 36 mg/I
C6H5FeO7.xH2O 6 mg/I
H3BO3 2.9 mg/I
MnCl2.4H2O 1.81 mg/I
ZnCl2 0.11 mg/I
CuSO4.5H2O 0.08 mg/I
(NH4)6Mo7O24.4H2O 0.018 mg/I

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10E4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water (tap water purified by reverseosmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:

NH4CI 15 mg/I
MgCl2.6H2O 12 mg/I
CaCl2.2H2O 18 mg/I
MgSO4. 7H2O 15 mg/I
KH2PO4 1.6 mg/I
FeCl3.6H2O 80 μg/l
Na2EDTA.2H2O 100 μg/l
H3BO3 185 μg/I
MnCl2.4H2O 415 μg/I
ZnCl2 3μg/l
CoCl2.6H2O 1.5 μg/I
CuCl2.2H2O 0.01 μg/l
Na2MoO4.2H2O 7 μg/l
NaHCO3 50 mg/I
Hardness (Ca+Mg} 0.24 mmol/I (24 mg CaCO3/l)
pH 8.3 ±0.2


ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

24h and 72h

Hardness:

0.24 mmol/I (24 mg CaCO3/l)

Test temperature:

between 23.6 and 23.9°C

pH:

8.0 - 8.2

Dissolved oxygen:

Not specified

Salinity:

Not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 50 ml of test medium
- Initial cells density: An initial cell density of 0.98x10E4 cells/ml (instead of 1.0x104 cells/ml prescribed by the protocol)

Replicates
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Replicates of the highest concentration without algae: 2
- 1 additional replicate of the blank-control and the test concentration for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: Same

OTHER TEST CONDITIONS
- Photoperiod and Light intensity:
Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 75 to 104 μE.m-2.s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Effect parameter Concentration Setafix X 11342 (μg/I)
Initial measured Average exposure
Observed NOEC 42 11
72h-E(B)C50 > 94 > 14
72h-E(R)C50 > 94 > 14

In general, EC50 values were above water solubility.
The E(B)C50 is the concentration of test substance that results in a 50% inhibition of total cell growth relative to the control.
The E(R)C50 is the concentration of test substance that results in a 50% reduction in growth rate relative to the control.
NOEC is the highest concentration tested at which the measured parameter(s) show (s) no significant effect on algal growth relative to control values.

TEST CONCENTRATIONS
- Limit Test: Measured Test Substance Concentrations:
See details on results


ADDITIONAL TEST
An additional test was performed in order to determine if the observed toxicity was caused by the test substance itself or by the impurities. Test conditions were comparable to those in the limit test with the following exceptions. Three replicates per concentration were exposed to a nominal concentration of 8.8 μg/I, to filtrates prepared at loading rates of 10, 50 and 100 mg/I, to dilutions containing 10 and 50% of the filtrate at 100 mg/I and to a blank-control. Light intensity varied between 75 and 105 μIE.m-2.s-1. Duplicate samples for analysis were taken from all concentrations and the blank··control at the start of the test, after 24 hours and at the end of the test. Vessels without algae were not included in this test.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 94 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.94 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Dose descriptor:

NOEC

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: cell growth inhibition and growth rate reduction

Remarks on result:

other: Duration for NOEC value not specified

Dose descriptor:

NOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell growth inhibition and growth rate reduction

Remarks on result:

other: Duration for NOEC value not specified

Details on results:

LIMIT TEST

Measured Test Substance Concentrations:

The study started with a limit test, exposing exponentially growing algal cultures to a filtrate prepared at a loading rate of 1100 mg/I and to a blank-control.
At the start of the test, the actual test concentration in the filtrate was 97 μg/I. This concentration decreased to 7.2 μg/I within the first 24 hours and further to 3.1 μg/I, i.e. below the water· solubility (8.8 μg/I), during the remainder of the test period.
The measured concentration in the samples taken from the solutions without algae showed a lower decrease, indicating that adsorption by algal cells was at least partly responsible for the decrease observed in the presence of algae. Based on these results, the average exposure concentration was calculated to be 12 μg/I, being slightly higher than the water solubility of the test substance.

Inhibition of cell growth and reduction of growth rate:

- Cell growth and growth rate were statistically significantly reduced upon exposure to Setafix X 11342.

- It was clear that the EC50 values for both cell growth and growth rate were above water splubility of the main component in Setafix X 11342. A NOEC could not be determined. However, since impurities were found in high amounts in the chromatograms, it was hypothesized that the observed effects may have be·en caused by these impurities. Moreover, it was not clear if the test substance was toxic at its water solubility. Therefore, an additional test was performed exposing algae to a target concentration equal to the water solubility, and using two concentration series with differing methods of preparation.

ADDITIONAL TEST

Measured Test Substance Concentrations:

- At the start of the test, the actual test concentration at nominal 8.8 μg/I was 7.3 μg/l. The initial concentrations measured in the undiluted filtrates prepared at 100, 50 and 10 mg/I were respectively 45, 94 and 42 μg/l.
- The difference between the actual concentrations measured in the filtrates prepared at 100 and 50 mg/I, as well as the similarity of those measured inthe filtrates prepared at 100 and 10 mg/I may be explained by the extremely low solubility of the test substance and is an inherent c:onsequence of the method of preparation. Dilutions of the filtrate prepared at too mg/I contained actual concentrations that were in general agreement with what was expected.
- In accordance with what was found in the limit test, all concentrations decreased during the test, even those that were at the level of water solubility at the start of the test. This indicates that the solubility of the test substance in test medium is even lower than that in water. At the end of the
test, all test concentrations had decreased below the limit of detection (i.e. < 4 μg/I) and were below the water solubility of 8.8 μg/I.

Inhibition of cell growth and reduction of growth rate:

- Inhibition of cell growth increased with increasing concentration of Setafix X 11342, but also with increasing presence of impuri1ties.
- In the solution aimed at water solubility (nominal 8.8 μg/1), cell growth was not inhibited
- Statiistically significant inhibition of cell growth was found in the filtrates prepared at 50 and 100 mg/I
- Cell growth inhibition was higher in the filtrate at 100 mg1/I than in the filtrate at 50 mg/I, even though initial and average exposure concentrations in th4e filtrate at 100 mg/I were lower than those in the filtrate at 50 mg/I
- This difference in response may at least partly be caused by the presence of impurities, which were more abundant in the filtrate at 100 mg/I than in the filtrate at 50 mg/I. Similarly, responses observed in the filtrates at 10 and 50 mg/I were higher compared to those in the dilutions containing 10 and 50'3 of the filtrate prepared at 100 mg/I.
- Considering the initial and average exposure concentrations, the role of the impurities is much less evident than in the comparison between filtrate at 50 and 100 mg/I, but cannot be excluded, since impurities were more abundant in the filtrates than in the dilutions.
- Growth rates were in the range of the controls in the solution aimed at the water solubility (nominal 8.8 μg/I), in the filtrate prepared at 10 mg/I and in the dilutions of the filtrate prepared aT 100 mg/I. Reduction of growth rate decreased as exposure progressed.
- Statistically significant reduction of growth rate was found in the filtrates prepared at 50 and 100 mg/I
- Growth rate reduction was higher in the filtrate prepared at 100 mg/I compared to that prepared at 50 mg/I, despite of lower initial and average exposure concentrations in the filtrate at 100 mg/I. This may be caused by the presence of impurities, as discussed for cell growth inhibition.
- It should be noted that both c13ll growth and growth rate were not affected in solutions where the initial concentration approach,ed the water solubility (i.e., nominal 8.8 μg/I and dilution containing 10% of the filtrate prepared at 1 00 mg/I), indicating that the main component in Setafix X 11342
is not toxic to algae.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- Under the conditions of the mference study with Selenastrum capricornutum, potassium dichromate inhibited cell growth and reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/I and higher.

- The EC50 for cell growth inhibition [E(B)C50: 0-72h] was 0.86 mg/I with a 95% confidence interval ranging from 0.57 to 1.3 mg/I .. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/I. Hence, the E(B)C50: 0-72h for the present batch corresponds with this range.

- The EC50 for growth rate reduction [E(R)C50: 0-72h] was 1.5 mg/I with a 95% confidence interval ranging from 1 .1 to 2.1 mg/I. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/I. Hence, the E(R)C50: 0-72h for the present batch corresponds with this range.

Validity criteria fulfilled:

yes

Remarks:

The test met the validity criteria as prescribed by the protocol and was considered valid

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, Setafix X 11342 inhibited cell growth and reduced growth rate of this fresh water algae species significantly in filtrates prepared at loading rates of 50 and 100 mg/l. This could at least partly be attributed to toxicity caused by the impurities present in the test substance. Cell growth and growth rate were not affected in solutions where the initial concentration approximated the water solubility of the main component.

The EC50 for cell growth and growth rate were above water solubility.

The NOEC for both cell growth inhibition and growth rate reduction was 0.042 mg/l when based on initial concentrations, and 0.011 mg/l when based on average exposure concentrations.

Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with Setafix X 11342.

The study procedures described in this report were based on the ISO International Standard 8692, 1989. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992 and the OECD guideline No. 201, 1984.

The batch of Setafix X 11342 tested was an off-white to yellowish solid with a purity of >95% and the substance was not completely soluble in test medium at the concentrations tested.

In the limit test, preparation of the test solution started with a stock solution at a loading rate of 100 mg/l applying 3 days of magnetic stirring to reach maximum solubility in test medium. Subsequently, the dispersion was filtered over a rough paper filter to remove the larger undissolved particles. The final solution was clear and colourless.

In the additional test, preparation of test solutions started with four stock solutions. One stock was prepared in DMSO to reach a concentration of 8.8 μg/l in test medium. In addition, three filtrates were prepared as in the limit test, at loading rates of 10, 50 and 100 mg/l. Finally, the filtrate at 100 mg/l was diluted twice and ten-fold. All test solutions were clear and colourless.

The study started with a limit test, exposing exponentially growing algal cultures to a filtrate prepared at a loading rate of 1100 mg/l and to a blank-control. The total test period was 72 hours. Samples for analysis were tak;en at the start of the test, after 24 hours and at the end of the test. Cell growth was inhibited by 39%, while growth rate was reduced by 14%. Both responses were statistically significant. The initial concentration in the filtrate was 9.7 μg/l. This concentration decreased to 7.2 μg/l within the first 24 hours and further to 3.1 μg/l, i.e. below the water solubility (8.8 μg/l), during the remainder of the test period. The average exposure concentration was 1.2 mg/l, being slightly higher than the water solubility of the test substance. Additional peaks were observed in the chromatograms. These peaks were assumed to derive from the impurities of the test substance, which appeared to be more soluble in test medium than the major component, and as such may have been (partly) responsible for the observed effects.

An additional test was performed to determine the toxicity of the test substance at its water solubility. In addition, two series of concentrations were tested in order to assess whether the impurities were responsible for the observed effects. Three replicates were exposed per concentration and a blank-control. Concentrations were nominal 8.8 μg/l, filtrates prepared at loading rates of 10, 50 and 100 mg/l, and dilutions containing 10 and 50% of the filtrate at 100 mg/l. Samples for analysis were taken from all concentrations and the blank-control at the start of the test, after 24 hours and at the end of the test.

At the start of the test, the actual test concentration at nominal 8.8 μg/l was 7.3 μg/l. The initial concentrations measured in the undiluted filtrates prepared at 100, 50 and 10 mg/l were respectively 45, 94 and 42 μg/l. Dilutions of the filtrate prepared at 100 mg/l contained actual concentrations that were in gemeral agreement with what was expected.

All concentrations decreased during the test, even those that were at the level of water solubility at the start of the test, indicating that the solubility of the test substance in test medium is even lower than that in water. At thta end of the test, all test concentrations had decreased below the limit of detection and were bellow the water solubility of 8.8 μg/l.

Impurities were observed in all chromatograms, except for the solution prepared aimed at water solubility (nominal 8.8 μg/l). These impurities increased with concentration in the dilution series of the filtrate prepared at 100 mg/I, as well as in the series of filtrates prepared at increasing concentrations. Furthermore, concentrations of impurities were slightly higher in filtrates prepared at 10 and 50 mg/l than in dilutions containing 10 and 50% of the filtrate prepared at 100 mg/l, respectively. Impurities decreased in time, and were at or just above the noise level at the end of the test.

The test met the validity criteria as prescribed by the protocol and was considered valid.

Setafix X 11342 inhibited cell growth and reduced growth rate of this fresh water algae species significantly in filtrates prepared at loading rates of 50 and 100 mg/l. This could at least partly be attributed to impurities. Cell growth and growth rate were not affected in solutions where the initial concentration approximated the water solubility of the main component.

The EC₅₀ for cell growth and growth rate were above water solubility.

The NOEC for both cell growth inhibition and growth rate reduction was 0.042 mg/I when based on initial concentrations, and 0.011 mg/l when based on average exposure concentrations.

Description of key information

Setafix X 11342 inhibited cell growth and reduced growth rate of this fresh water algae species significantly in filtrates prepared at loading rates of 50 and 100 mg/l. This could at least partly be attributed to impurities. Cell growth and growth rate were not affected in solutions where the initial concentration approximated the water solubility of the main component.
The EC50 for cell growth and growth rate were above water solubility.
The NOEC for both cell growth inhibition and growth rate reduction was 0.042 mg/l when based on initial concentrations, and 0.011 mg/l when based on average exposure concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.011 mg/L

Additional information

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with Setafix X 11342.

The study procedures described in this report were based on the ISO International Standard 8692, 1989. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992 and the OECD guideline No. 201, 1984.

The batch of Setafix X 11342 tested was an off-white to yellowish solid with a purity of >95% and the substance was not completely soluble in test medium at the concentrations tested.

In the limit test, preparation of the test solution started with a stock solution at a loading rate of 100 mg/l applying 3 days of magnetic stirring to reach maximum solubility in test medium. Subsequently, the dispersion was filtered over a rough paper filter to remove the larger undissolved particles. The final solution was clear and colourless.

In the additional test, preparation of test solutions started with four stock solutions. One stock was prepared in DMSO to reach a concentration of 8.8 μg/l in test medium. In addition, three filtrates were prepared as in the limit test, at loading rates of 10, 50 and 100 mg/l. Finally, the filtrate at 100 mg/l was diluted twice and ten-fold. All test solutions were clear and colourless.

The study started with a limit test, exposing exponentially growing algal cultures to a filtrate prepared at a loading rate of 1100 mg/l and to a blank-control. The total test period was 72 hours. Samples for analysis were taken at the start of the test, after 24 hours and at the end of the test. Cell growth was inhibited by 39%, while growth rate was reduced by 14%. Both responses were statistically significant. The initial concentration in the filtrate was 9.7 μg/l. This concentration decreased to 7.2 μg/l within the first 24 hours and further to 3.1 μg/l, i.e. below the water solubility (8.8 μg/l), during the remainder of the test period. The average exposure concentration was 1.2 mg/l, being slightly higher than the water solubility of the test substance. Additional peaks were observed in the chromatograms. These peaks were assumed to derive from the impurities of the test substance, which appeared to be more soluble in test medium than the major component, and as such may have been (partly) responsible for the observed effects.

An additional test was performed to determine the toxicity of the test substance at its water solubility. In addition, two series of concentrations were tested in order to assess whether the impurities were responsible for the observed effects. Three replicates were exposed per concentration and a blank-control. Concentrations were nominal 8.8 μg/l, filtrates prepared at loading rates of 10, 50 and 100 mg/l, and dilutions containing 10 and 50% of the filtrate at 100 mg/l. Samples for analysis were taken from all concentrations and the blank-control at the start of the test, after 24 hours and at the end of the test.

At the start of the test, the actual test concentration at nominal 8.8 μg/l was 7.3 μg/l. The initial concentrations measured in the undiluted filtrates prepared at 100, 50 and 10 mg/l were respectively 45, 94 and 42 μg/l. Dilutions of the filtrate prepared at 100 mg/l contained actual concentrations that were in general agreement with what was expected.

All concentrations decreased during the test, even those that were at the level of water solubility at the start of the test, indicating that the solubility of the test substance in test medium is even lower than that in water. At the end of the test, all test concentrations had decreased below the limit of detection and were bellow the water solubility of 8.8 μg/l.

Impurities were observed in all chromatograms, except for the solution prepared aimed at water solubility (nominal 8.8 μg/l). These impurities increased with concentration in the dilution series of the filtrate prepared at 100 mg/I, as well as in the series of filtrates prepared at increasing concentrations. Furthermore, concentrations of impurities were slightly higher in filtrates prepared at 10 and 50 mg/l than in dilutions containing 10 and 50% of the filtrate prepared at 100 mg/l, respectively. Impurities decreased in time, and were at or just above the noise level at the end of the test.

The test met the validity criteria as prescribed by the protocol and was considered valid.

Setafix X 11342 inhibited cell growth and reduced growth rate of this fresh water algae species significantly in filtrates prepared at loading rates of 50 and 100 mg/l. This could at least partly be attributed to impurities. Cell growth and growth rate were not affected in solutions where the initial concentration approximated the water solubility of the main component.

The EC₅₀for cell growth and growth rate were above water solubility.

The NOEC for both cell growth inhibition and growth rate reduction was 0.042 mg/l when based on initial concentrations, and 0.011 mg/l when based on average exposure concentrations.