Benzene, 2-(3-chloropropoxy)-1-methoxy-4-nitro-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2009 - 22 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed in a GLP laboratory to OECD guidelines without deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

1. Temperature outside the ± 2°C range during the final test period (i.e. 2.1°C). Temperature remained within 21-24°C and algal cells showed exponential growth. 2. The cell density was measured wit a cuvette instead of a probe.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(3-chloropropoxy)-1-methoxy-4-nitrobenzene

EC Number:

618-883-1

Cas Number:

92878-95-0

Molecular formula:

C10H12ClNO4

IUPAC Name:

2-(3-chloropropoxy)-1-methoxy-4-nitrobenzene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: WAY-188471
Molecular formula: C10H12ClNO4
Molecular weight: 245.66
CAS Number: 92878-95-0
Description: Slightly yellow powder
Test substance storage: At room temperature protected from light
Stability under storage conditions: Stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2 ml sample volumes were obtained for all concentrations.
- Sample storage conditions before analysis: Samples we stored in a freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The batch of WAY-188471 tested was a slightly yellow powder with a purity of 99.8% and not completely soluble in test medium at the concentrations tested. Note that weighing was performed in yellow light and preparation was performed using dimmed light conditions.
Preparation started with a loading rate of 100 mg/l applying a two-day period of magnetic stirring. The obtained solution containing undissolved material was filtered through a membrane filter (Whatman, 0.45 μm). The lower concentrations were prepared by subsequent dillutions in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.


- Controls:
Test medium without test substance or other additives. Six (6) replicates of the blank control were used.

- Evidence of undissolved material:
No data.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source: In-house laboratory culture.
- Age of inoculum: 3 days
- Method of cultivation: Growth medium was inoculated 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 21-24°C.

ACCLIMATION
- Acclimation period: 3 days prior to testing.
- Culturing media and conditions: Same as test

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 22.3°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 21.9 and 24.0°C. Temperature remained within the limits prescribed by the protocol (21-24°C) but varied slightly outside ± 2°C.

pH:

pH 8.1 ± 0.2

Dissolved oxygen:

No data.

Salinity:

no data, freshwater was used.

Nominal and measured concentrations:

Analyses showed initial measured concentrations of 0.41, 0.95, 2.0, 4.3, and 9.2 mg/l in the samples taken from respectively 4.6, 10, 22, 46, and 100% of the filtrate prepared at a loading rate of 100 mg/l. These concentrations remained stable during the test period (98-104% of initial). Based on these observations the effect parameters were based on the initial concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Aeration: The suspensions were continuously aerated.
- Initial cells density: 1 x 104 cells/ml.
- Control end cells density: 1 x 104 cells/ml.
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes, M2 test medium according to OECD 201 was used.

OTHER TEST CONDITIONS
- Photoperiod:
- Light intensity and quality: No illumination during the first 24 hours of exposure. Thereafter, illumination was continuous using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 84 to 100 E.m-2.s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: spectrophotometric measurement at 720 nm.

TEST CONCENTRATIONS
- Test concentrations: 0.41, 0.95, 2.0, 4.3, and 9.2 mg/l
- Results used to determine the conditions for the definitive study: Cell density

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: >4.9 to <6.5

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield inhibition

Remarks on result:

other: >1.2 to <7.1

Any other information on results incl. tables

COMBINED LIMIT/RANGE-FINDING TEST

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 presents the percentages growth rate reduction and yield inhibition per concentration. Based on these results samples taken from the 0.45 µm filtered solution prepared at a loading rate of 100 mg/l and 10% of the filtrate were analysed. The initial concentrations were 0.80 and 7.9 mg/l. These concentrations remained stable during the test period (105-108% of initial, see also Table 2 of the appended Analytical Report). Therefore, the expected EC50 for growth rate reduction and yield inhibition was between 0.80 and 7.9 mg/l.

Table 1 Mean cell densities (x104 cells/ml) during the combined limit/range-finding test Initial conc. WAY-188471 (mg/l) Exposure time (hours) 0 24 48 72 96 control 1.0 1.0 4.3 30.8 154.3 0.0079* 1.0 1.0 3.1 27.0 137.7 0.079* 1.0 1.0 4.6 28.4 143.8 0.80 1.0 1.0 4.9 28.9 131.9 7.9 1.0 1.0 1.5 1.2 1.1

Table 2: Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test Initial conc. WAY-188471 (mg/l) Mean growth rate Yield (0-96 h) µ (24-96 h) Reduction (%) x104cells/ml Inhibition (%) control 0.06996   153.33   0.0079* 0.06839 2.2 136.68 10.9 0.079* 0.06897 1.4 142.81 6.9 0.8 0.06773 3.2 130.9 14.6 7.9 0.00158 97.7 0.13 99.9

FINAL TEST

Table 3 shows mean cell densities measured at 24-hour intervals at the different concentrations of WAY-188471.

Table 3: Mean cell densities (x 104 cells/ml) during the final test Initial conc. WAY-188471 (mg/l) Exposure time (hours) 0 24 48 72 96 control 1 1 5.6 29.6 138.2 0.41 1 1 6 33.6 150.7 0.95 1 1 5.9 27.7 129.5 2 1 1 6.2 27.4 123.6 4.3 1 1 5.9 13.1 21.7 9.2 1 1 3.7 3.3 3.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The study generally met the acceptability criteria prescribed by the protocol and was considered valid.

Conclusions:

WAY-188471 reduced growth rate and inhibited the yield of this fresh water algae species significantly at an initial concentration of 4.3 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-96h) was 5.6 mg/l with a 95% confidence interval ranging from 4.9 to 6.5 mg/l.
The EC50 for yield inhibition (EYC50: 0-96h) was 3.0 mg/l with a 95% confidence interval ranging from 1.2 to 7.1 mg/l.
The NOEC for growth rate reduction and yield inhibition was 2.0 mg/l.