1,3-dichlorobenzene

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:

24 January 1986 to 28 June 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to GLP and in accordance with an older version of OECD Guideline 201 (1984). The quality of the study is considered suitable for hazard assessment in accordance with modern requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(1984)

Deviations:

not specified

GLP compliance:

yes

Remarks:

This study was conducted in accordance with the Japanese Ministry of the Environment “Good Laboratory Practice for Ecological Impact Studies”

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At all concentrations, test solutions (not inoculated with algae) prepared for the analysis samples were analysed at the beginning of exposure. At the end of exposure, test solutions were collected from test vessels (3 replicates) in each concentration group in approximately equal amounts and analysed.
- Sampling method: When collecting the solutions, a 0.45 µm filter was connected to a syringe to remove the algae. Two 20 mL samples (two 40 mL samples for the control) were collected in a centrifuge tube, for subsequent extraction (see details on analytical method)
- Sample storage conditions before analysis: Not specified

Vehicle:

yes

Remarks:

1:3 mixture of hardened castor oil (HCO-40):ethanol

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The required amount of test substance was weighed using an electronic balance, this was made up to volume with a 1:3 mixture of hardened castor oil (HCO-40):ethanol as a dissolution aid to prepare a standard stock solution. The volume required for each nominal concentration was weighed out from this standard stock solution, and was made up to volume with the dissolution aid (the dissolution aid concentration was constant for each concentration). By adding fixed volumes of these solutions, test solutions were prepared at the nominal concentrations of 1.0, 3.2, 5.6, 7.5, and 10 mg/L. The test solutions were sealed immediately after preparation.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphiodocelis supcapitata (defined in study report as Selenastrum capricornutum due to age of study)
- Strain: NIES-35
- Source (laboratory, culture collection): In-house culture
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Asceptic

ACCLIMATION
- Acclimation period: The alga used for the test was precultured for three days before start of exposure under the same conditions as the test conditions.
- Culturing media and conditions (same as test or not): Not specified
- Any deformed or abnormal cells observed: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Default guideline exposure duration

Post exposure observation period:

No

Test temperature:

Daily Temperature in the Incubation Chamber during 72-hour Exposure:
0h: 23.4 ºC
24h: 23.0 ºC
48h: 23.5 ºC
72h: 23.1 ºC
Average ± SD = 23.3 ± 0.24

pH:

pH Values at 0h and 72h Exposure
Control - 0h: 8.0; 72h: 10.5 ± 0.06
Solvent Control 0h: 8.0; 72h: 10.37 ± 0.06
1.0 mg/L - 0h: 8.0; 72h: 10.47 ± 0.06
3.2 mg/L - 0h: 8.0; 72h: 10.43 ± 0.06
5.6 mg/L - 0h: 8.0; 72h: 10.13 ± 0.06
7.5 mg/L - 0h: 8.0; 72h: 9.47 ± 0.06
10 mg/L - 0h: 8.0; 72h: 7.23 ± 0.06

Nominal and measured concentrations:

Nominal Concentrations: 1.0, 3.2, 5.6, 7.5 and 10 mg/L plus control
Geometric Mean Measured Concentrations: 0.71, 2.16, 3.61, 5.02, 6.33 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass Erlenmeyer flask (with Teflon seal stopcock)
- Type: closed
- Fill volume: 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 1 × 10e4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not specified
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH: 0 and 72h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6400−8800 lux
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter
- Chlorophyll measurement: Not applicable
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: Not specified
- Results used to determine the conditions for the definitive study: Yes

CULTURING APPARATUS
-Details on culturing apparatus used: Shake culture (100 rpm)

Reference substance (positive control):

yes

Remarks:

The 72-hour growth inhibition concentration (EbC50) of the reference substance (potassium dichromate, guaranteed reagent, Lot No. KCE6888, Wako Pure Chemical Industries) was 0.86 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.9 mg/L

95% CI:

>= 1.6 - <= 20.3

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.2 mg/L

95% CI:

>= 2.4 - <= 32

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2.4 ~ 32 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not specified
- Unusual cell shape: Not specified
- Colour differences: Not specified
- Flocculation: Not specified
- Adherence to test vessels: Not specified
- Aggregation of algal cells: Not specified
- Other:
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Table 1. Measured Concentrations of m-Dichlorobenzene during a 72-hour Algae Exposure

Nominal Concentration (mg/L)	Measured Concentration (mg/L)
	0 hour	% Nominal	72 hour	% Nominal	Geometric Mean Measured Concentration
Control	<0.01	-	<0.01	-	0
Solvent Control	<0.1	-	<0.1	-	0
1.0	1.0	100	0.5	50	0.71
3.2	3.1	97	1.5	47	2.16
5.6	5.0	89	2.6	46	3.61
7.5	7.4	99	3.4	45	5.02
10	8.9	89	4.5	45	6.33

Table 2. Growth Inhibition following 72-hour Algae Exposure

Concentration		Area	Inhibition (%)	Rate	Inhibition (%)	Rate	Inhibition (%)
mg/L	No.	A(0-72h)	I^(0-72h)	µ (24-48h)	Im(24-48h)	µ(24-72h)	Im(24-72h)
Control	1	12534000	-	0.0721	-	0.0515	-
	2	15070560	-	0.0718	-	0.0481	-
	3	14963760	-	0.0791	-	0.0490	-
	Average	14189440	NA	0.0743	NA	0.0495	NA
Solvent Control	1	16021200	-	0.0808	-	0.0530	-
	2	15326880	-	0.0841	-	0.0517	-
	3	13979520	-	0.0716	-	0.0478	-
	Average	15109200	-6.5	0.0788	-6.1	0.0508	-2.6
1.0	1	13999200	-	0.0650	-	0.0475	-
	2	14350080	-	0.0739	-	0.0460	-
	3	15166800	-	0.0891	-	0.0513	-
	Average	14505360	-2.2	0.0760	-2.3	0.0483	2.4
3.2	1	14799360	-	0.0788	-	0.0514	-
	2	14234160	-	0.0799	-	0.0500	-
	3	13052640	-	0.0800	-	0.0475	-
	Average	14028720	1.1	0.0796	-7.1	0.0496	-0.2
5.6	1	8564160	-	0.0699	-	0.0471	-
	2	8316240	-	0.0638	-	0.0461	-
	3	8211840	-	0.0694	-	0.0439	-
	Average	8364080	41.1	0.0677	8.9	0.0457	7.7
7.5	1	4529520	-	0.0526	-	0.0252	-
	2	4116480	-	0.0769	-	0.0317	-
	3	4719840	-	0.0628	-	0.0256	-
	Average	4455280	68.6	0.0641	13.7	0.0275	44.4
10	1	3230160	-	0.0577	-	0.0203	-
	2	3295920	-	0.0655	-	0.0193	-
	3	2803440	-	0.0690	-	0.0189	-
	Average	3109840	78.1	0.0644	13.3	0.0195	60.6

 

 

 

 

 

Validity criteria fulfilled:

yes

Remarks:

Validity criteria in accordance with OECD 201 (1984) were met

Conclusions:

The 72h ErC50 (growth rate) was 5.9 mg/L (GMM) and the 72h NOErC (growth rate) was 2.1 mg/L.

Executive summary:

In a study conducted in accordance with OECD 201 (1984), and in accordance with GLP, the growth inhibition concentration (EC50) and no-observed-effect concentration (NOEC) of the test item against the unicellular green algae (Selenastrum capricornutum) was determined.  Based on the results of a range-finding test, the study was conducted at 5 test concentrations of 1.0, 3.2, 5.6, 7.5, and 10 mg/L, including an untreated and vehicle control.  Test item concentrations were determined at 0 and 72h during the test.  All test and control vessels were inoculated with 1x10e4 cells/mL, and subsequent cell concentrations were determined in each vessel at 24, 48 and 72h.  The pH of each vessel and temperature of the incubation chamber were monitored during the test.  Due to a reduction in test concentration analysed during the test, key results were based on geometric mean measured concentrations. The mean increase in cell concentration after 72 hours of incubation was 55-fold in the control and the vehicle control.  In the 1.0 and 3.2 mg/L groups, growth was around 50-fold, which was similar to the control group, but at 5.6 and 7.5 mg/L growth fell to 32-fold and 11-fold, respectively, while growth at the highest concentration of 10 mg/L was very low at 6-fold. The 72h ErC50 (growth rate) was 5.9 mg/L (GMM) and the 72h NOErC (growth rate) was 2.1 mg/L.

Description of key information

72h ErC50 = 5.9 mg/L (GMM), 72h NOErC = 2.1 mg/L (GMM); OECD 201 (1984); MITI, 1995

Key value for chemical safety assessment

EC50 for freshwater algae:

5.9 mg/L

EC10 or NOEC for freshwater algae:

2.1 mg/L

Additional information

OECD 201 (MITI, 1995)

In a study conducted in accordance with OECD 201 (1984), and in accordance with GLP, the growth inhibition concentration (EC50) and no-observed-effect concentration (NOEC) of the test item against the unicellular green algae (Selenastrum capricornutum) was determined.  Based on the results of a range-finding test, the study was conducted at 5 test concentrations of 1.0, 3.2, 5.6, 7.5, and 10 mg/L, including an untreated and vehicle control.  Test item concentrations were determined at 0 and 72h during the test.  All test and control vessels were inoculated with 1x10e4 cells/mL, and subsequent cell concentrations were determined in each vessel at 24, 48 and 72h.  The pH of each vessel and temperature of the incubation chamber were monitored during the test.  Due to a reduction in test concentration analysed during the test, key results were based on geometric mean measured concentrations. The mean increase in cell concentration after 72 hours of incubation was 55-fold in the control and the vehicle control.  In the 1.0 and 3.2 mg/L groups, growth was around 50-fold, which was similar to the control group, but at 5.6 and 7.5 mg/L growth fell to 32-fold and 11-fold, respectively, while growth at the highest concentration of 10 mg/L was very low at 6-fold. The 72h ErC50 (growth rate) was 5.9 mg/L (GMM) and the 72h NOErC (growth rate) was 2.1 mg/L.