Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The analogue substance which shares the same functional groups with the target substance also has comparable values for the relevant molecular properties. The results obtained can be used for the read-across approach. Test method according to OECD 201. GLP 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:

- Concentrations: Water samples were taken from the control and 2.2 mg/L test group (r1-r3 pooled, r4-r6 pooled) at 0 and 72 hours for quantitative analysis.
- Sampling method: A volume of test sample was extracted with dichloromethane (3x30mL). The combined extracts were made to volume in dichloromethane (100 mL final volume) to give a final theoretical concentration of 6.6 mg/L.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored approximately at 0ºC, sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was considered difficult substance due to his poorly water solubility, media preparation trials were conducted. The use of a saturated solution was considered the most appropriate method.
An amount of test material (550 mg) was dispersedin 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of 21ºC for a period of 24 hours. After 24 hours large particles of undissolved material were removed by filtration through Postlip BW/S filter (10 µm retention size) prior to centrifugation at 40000 g for 30 minutes to give the satured solution with nominal concentration of 2.2 mg/L. (based on the results of the media preparation trials)
- Eluate: Reconstituted water.
- Controls: Negative solvent (water) control group.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): During the range-finding test it was noted that some undissolved material remained in the supernatant after centrifugation it was therefore considered appropriate to filter the supernatant through Postlip filter paper (10 µm retnetion size)prior to centrifugation in the definitive test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: CCAR 276/20
- Source (laboratory, culture collection): Culture collection of Algae and Protozoa (CCAP), institute for Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.
- Age of inoculum (at test initiation): Preculture conditions gave an algal suspension in log phase growth characterized by a densityof 2.4 x 1000000 cells per mL.
- Method of cultivation: The culture was mantained in the laboratory at a temperature of 21 ± 1 ºC under continous ilumination (approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not required.

Test temperature:

24 ± 1

pH:

7.3 - 8.6

Nominal and measured concentrations:

Nominal concentration: 2.2 mg/L
Geometric mean measured test concentration: 0.0275 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type: The flasks were plugged with polyurethane foam bungs.
- Material, size, headspace, fill volume: 250 mL, glass.
- Initial cells density: Mean control: 9770 cells per mL; Mean test group: 11200 cells per mL
- Control end cells density: Mean control 72h: 552000 cells per mL.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: AAP-medium (US. EPA) with some differences:
MgCl2 6H2O: 12.164 mg/L
CaCl2 2H2O: 4.41 mg/L
MgSO4 7H2O: 14.7mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 15.0 mg/L
H3BO3: 0.1855 mg/L
MnCl2 4H2O: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl3 6 H2O: 0.159 mg/L
CoCl2 6H2O: 0.00143 mg/L
Na2MoO4 2H2O: 0.00726 mg/L
CuCl2 2H2O: 0.000012 mg/L
Na2EDTA 2H2O: 0.30 mg/L
Na2SeO3 5H2O: 0.000010 mg/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga optima 15 +or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: The culture medium was sterilised by 0.2 µm membrane filtration.
- Adjustment of pH: Yes, culture medium was adjusted to pH 7.5 ± 0.1 with 0.1 N NaOH or HCl
- Photoperiod: Continuous ilumination
- Light intensity and quality: approximately 7000 lux.
- Other: Constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and cell densities were determined using a Coulter® Multisizer Particle Counter
- Other: pH was recorded at initiation and at the end. Temperature was recorded daily.

TEST CONCENTRATIONS
A limit test was conduced in the main part of the study with the saturated solution (2.2 mg/L nominal, obtained in media preparation trials)
- Justification for using less concentrations than requested by guideline: The test substance was considered a difficult substance because his poorly water solubility. (OECD guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD 2000). Media preparation trials were undertaken, the method of the saturated solution resulted to be the most appropriate.
- Range finding study: A preliminary range-finding test was performed with the saturated solution (nominal from media preparation trial: 2.2 mg/L) at 100, 10 and 1 %.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 1, 10 and 100% v/v saturated solution.

Reference substance (positive control):

not required

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

> 0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

- Exponential growth in the control: yes, the cell concentration of the control cultures increased by a factor of 57 during the test.
- Observation of abnormalities (for algal test): No microscopically abnormalities at 72 hours in any of the control of test cultures.
- Colour differences: At the start of the test all controls and test cultures were obserbed to be colourless solutions. After the 72-hour test period all control and test cultures were observed to be green dispersions.
- Any stimulation of growth found in any treatment: The test cultures (2.2 mg/L) showed a 7% of increase in growth compared to the controls.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: During the range-finding test it was noted that some undissolved material remained in the supernatant after centrifugation it was therefore considered appropriate to filter the supernatant through Postlip filter paper (10 µm retnetion size)prior to centrifugation in the definitive test.
- Effect concentrations exceeding solubility of substance in test medium: The study showed that there were no toxic effects at saturation.

Results with reference substance (positive control):

Not required.

Reported statistics and error estimates:

Statistical analysisof the area under the growth curve data was carried out for the control and all test concentrations using a t-Stundents test incorporating Barlett's test for homogeneity of variance (Sokal and Rohlf 1981).

Table 1. Cell densities and pH values in the definitive test

Nominal test concentration (mg/L)*		pH	Cell densities ** (cells per mL)				pH
		0h	0h	24 h	48h	72h	72h
Control	R1	7.5	10700	57100	76200	532000	8.4
	R2	7.5	10200	52700	73400	576000	8.4
	R3	7.5	8450	50900	78200	549000	8.4
	Mean		9770	53600	75900	552000
2.2	R1	7.3	12700	59100	72300	731000	8.6
	R2	7.3	11900	50500	76300	889000	8.6
	R3	7.3	10600	59300	76200	891000	8.6
	R4	7.3	10800	65300	79300	856000	8.6
	R5	7.3	10200	62100	81500	898000	8.6
	R6	7.3	11100	64600	82600	778000	8.6
	Mean		11200	60100	78000	840000

* Concentration determined from the media preparation trials conducted

**Cell densities represent the mean number of cells per mL calculated from the mean cell counts from 3 counts for each of the replicate flasks

R1 -R6: Replicates 1 to 6

Table 3 Inhibition of growth rate and biomass

Nominal concentration (mg/L)*	Area under curve at 72 h	% Inhibition	Growth rate (0-72 h)	% Inhibition
Control	9150000	-	0.056	-
2.2	12700000	[39]	0.060	[7]

* Concentration determined from media preparation trials conducted.

[Increase in growth compared to the controls]      

See the reporting format and data matrix attached.

Validity criteria fulfilled:

yes

Remarks:

The biomass of the control culture increase by a factor of 57 in the test period. The mean Coefficient of Variation for section-by-section specific growth rates in the control does not exceed 35%. The CoV of average specific growth does not exceed 10%

Conclusions:

The results of the limit test with a saturated solution (nominal 2.2mg/L and geometric mean measured 0.028 mg/L) indicate that the 48h-NOEC for the test substance is 0.028 mg/L based on the geometric mean measured concentration in Scenedesmus subspicatus.

Executive summary:

A freshwater Alga growth inhibition test was performed with a structural analogue of the registration substance called Nigrosine Base SAPL according to OECD Guideline 201 under GLP conditions with Scenedesmus subspicatus. The test substance fall into the category of difficult substance defined by the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures,OECD 2000. Media preparation trials were conducted, the saturated solution was considered the most appropriate method to prepare the medium at a saturated concentration of 2.2 mg/L. A rage-finding test was conducted, there where no immobilisation at 1, 10 and 100% of the concentration of the saturated solution. A limit main test was performed with with the saturated solution (2.2 mg/L nominal). 6 replicates using the saturated solution and 3 replicates of the control were conducted. The cultures were incubated on an orbital shaker under continuous illumination for 72 h. Growth was monitored daily by measuring the number of cells in each culture. An analytical monitoring was performed and validated. Due to the differences between the measured concentrations and the nominal concentration the following results are given with the geometric mean measured concentration: The EbC50 and ErC50 were > 0.028 mg /L and the NOEC was 0.028mg/L.

Description of key information

Key study: OECD 201. GLP study. The 72h-EC50 and the NOEC in green algae (basis for effect: growth inhibition rate) were determined to be >0.028 mg/L and 0.028 mg/L respectively based on the geometric mean measured concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.43 mg/L

EC10 or NOEC for freshwater algae:

0.027 mg/L

Additional information

Key study: Experimental results: An algae growth inhibition test was performed according to OECD Guideline 201 in green algae (Scenedesmus subspicatus). Based on growth inhibition rate, the 72h-EC50 and NOEC in green algae were determined to be greater than 0.028 and 0.028 mg/L respectively based on the geometric mean measured concentration under test conditions and up to the saturation concentration.