Reaction products of cyclohexylamine and 4-alkylaniline and 1,1'-methanediylbis(4-isocyanatobenzene)

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:

From 1996-03-01 to 1996-07-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although a guideline study (OECD 201 & EU Method C.3) conducted according to the principles of Good Laboratory Practice, the purity, batch number and expiry date of the test substance was not supplied and its stability under the test conditions was not determined. Also, verification of exposure concentrations during the study was not undertaken and the test results are based on the nominal concentrations used to prepare the test media.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

temperature of test area slightly exceeded maximum

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Substance type: technical material
- Physical state: a fine cream-coloured cohesive powder
- Storage condition of test material: at ambient temperature
- Other: Test concentrations quoted in the report refer to the test material as received; no allowance has been made for a purity of less than 100%

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium was prepared from an aqueous dispersion (nominally 111 mg/Litre) in which the test material, mixed with acetone, had been added to OECD medium; to aid dispersion it was subjected to twenty minutes of ultrasound treatment. This aqueous dilution was then shaken overnight in the dark before being centrifuged at 3000 rpm for 15 minutes to remove the bulk of the particulate material.
- Controls: algal suspension alone and a solvent control containing algal suspension and acetone.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L

Additional comment on medium preparation (information not presented in test report):

The test substance was multi-component and poorly soluble in water so the test media were prepared using techniques based on those employed for generating water accommodated fractions (WAFs). An auxiliary solvent was also used to aid dissolution and dispersion of the test substance. The physical methods employed were ultrasound and prolonged vigorous stirring. The formulation methods employed were designed to maximise the concentrations of the test substance in solution and therefore bioavailable to the test organisms. To avoid exposing the test organisms to large amounts of insoluble test material, the media used in the fish and Daphnia tests were left to equilibrate overnight before use and then the aqueous fraction was removed mid-vessel and used to fill the test vessels; for the algal study, the insoluble fraction of the test substance was removed by centrifugation. Although a concentrated stock was prepared for the fish test (to facilitate ultrasound treatment), the entire contents of the stock prepared for each batch of test medium was diluted to the required final volume, so the composition of the test mixture would not have been altered. Although the use of an auxiliary solvent may have an effect on the proportions of the different components dissolved, the amount used (0.1 mL/L) was very small and its impact is thought to have been negligible.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Freshwater Biological Association, Ferry House, Ambleside, Cumbria, UK.
- Age of inoculum (at test initiation): 2 months
- Method of cultivation: Liquid cultures for the test inoculum were established by inoculation of OECD medium (50 or 100 mL) with cells aseptically removed from slope cultures. Subsequently, subcultures were prepared from these liquid cultures by the aseptic transfer of aliquots into fresh OECD medium, which was then incubated for several days, until the required cell density had been achieved.

ACCLIMATION
- 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

Test temperature:

21.6 to 25.3 °C

pH:

7.2 to 8.4

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): loosely plugged with non-absorbent cotton wool
- Material, size, headspace, fill volume: 250 mL conical flasks, filled with 90 mL test medium
- Initial cells density: 1E+04 cells/mL
- Control end cells density: OECD control: 65.8-115 E+04 cells/mL; solvent control: 58.3-123 cells/mL
- No. of organisms per vessel: 1E+04 cellls per mL
- No. of vessels per concentration (replicates): 7(6 were incubated, the other used for temperature and pH at start)
- No. of vessels per control (replicates): 7 (6 were incubated, the other used for temperature and pH at start)
- No. of vessels per vehicle control (replicates): 7 (6 were incubated, the other used for temperature and pH at start)


GROWTH MEDIUM
- Standard medium used: yes (OECD)

OTHER TEST CONDITIONS
- Sterile test conditions: yes (aseptic conditions)
- Adjustment of pH: no
- Photoperiod: Continuous illumination was supplied by four, horizontallymounted, three-foot 30 watt "cool- white" fluorescent tubes.
- Light intensity and quality: 7700 lux (measurements taken with a Radiospares luxmeter during the definitive test showed 1900 - 1950 lux when a
single tube was illuminated).
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Haemocytometer (Improved Neubauer).

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/Litre
- Results used to determine the conditions for the definitive study: Several tests were conducted at nominal concentrations in the range 0.1 to 100 mg/L in which algal growth was adversely affected but the effects were neither concentration-related or reproducible. Following a test in which algae were exposed to centrifuged preparations containing test item it was concluded that the variability observed was due to particulate material in the test media. Therefore, 100mg/Litre chosen for definitive test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The median effect concentration (ErC50 or EbC50) could not be identified but it must be at least equal to the limit of aqueous solubility of the test material.

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The no-observed-effect concentration (NOEC) could not be identified but it must be at least equal to the limit of aqueous solubility of the test material.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Any stimulation of growth found in any treatment: No

Reported statistics and error estimates:

A statistical comparison of the average specific growth rates in control and test cultures was carried out using Dunnett's multicomparison test.
In this test, a multiple t-test compares each treatment with the control using a common estimate of experimental error, and then uses Dunnett's
t-Statistic tables to assess significance at the 95% level of probability.

Limit test - algal growth

The results of cell counts in control and test flasks are given in Table 1 below.

The average specific growth rates and biomass in control and test cultures are given in Table 2 below.

Exposure of Selenastrum capricornutum to an aqueous mixture of test item, derived from a preparation at an initial nominal concentration of 100 mg/Litre, did not result in a significant reduction of either the average specific growth rates or the mean biomass values compared to solvent control cultures (p > 0.05). Neither the median effect concentration (ErC50 or EbC50) nor the no-observed-effect concentrations could be identified but both must be at least equal to the limit of aqueous solubility of the test material.

Table 1.

Cell numbers in individual control and test cultures

Nominal test material conc. (mg/L)	Flask number	Cell numbers per mL (x10^4)
		24 hours	48 hours	72 hours
0 (OECD control)	1	6.38	33.3	86.3
	2	4.88	36.0	71.4
	3	5.13	29.5	74.1
	4	7.25	38.6	115
	5	5.50	32.3	65.8
	6	4.75	29.0	76.4
0 (solvent control)	8	6.00	31.4	71.3
	9	6.75	25.6	58.5
	10	3.63	27.8	60.3
	11	5.00	36.8	123
	12	5.00	23.9	58.6
	13	5.63	31.4	87.3
100	15	5.25	41.1	106
	16	4.38	25.9	59.1
	17	5.25	24.6	54.8
	18	5.00	33.1	97.5
	19	5.00	21.5	49.4
	20	4.50	22.8	51.0

Table 2.

Average specific growth rates and biomass (0-72 hours) in control and test cultures

Nominal test material conc. (mg/L)	Flask number	Average specific growth rate x10^-2	Mean x10^-2	Biomass x10^-4	Mean x10^-4
0 (OECD control)	1	6.191	6.088	1928	1848
	2	5.928		1778
	3	5.980		1660
	4	6.590		2420
	5	5.815		1637
	6	6.022		1667
0 (solvent control)	8	5.926	5.969	1693	1694
	9	5.651		1418
	10	5.694		1418
	11	6.684		2419
	12	5.654		1337
	13	6.207		1876
100	15	6.477	5.824 (2%)	2324	1569 (7%)
	16	5.666		1376
	17	5.561		1314
	18	6.361		2024
	19	5.417		1169
	20	5.461		1207

( ) : Percentage inhibition

Limit test - test environment

The temperature of the incubator ranged from 21.6 to 25.3°C during the test; although the temperature of the test area slightly exceeded that recommended by the test guidelines (maximum value 25°C), it is not considered to be significant nor to have affected the integrity of the test.

The temperature of the contents of control and test flasks ranged, at the start of the test, from 22.3 to 22.7°C and after 72 hours, from 23.7 to 24.0°C.

The pH of the contents of the flasks measured at the start of the test ranged betwen 7.8 and 8.4; after 72 hours pH ranged from 7.2 to 7.6.

On the day of preparation, the test medium was colourless with particulate material visible on its surface and on the base of the preparation flask. After centrifugation, the medium was colourless with a few particles of material on its surface.

Calculation of results

The effects of test item on the growth of Selenastrum cultures were determined by examining the growth rates of cultures (the rate of change in cell number with time) and biomass (the actual number of cells per volume of medium) as follows. Calculations were based on the the values given in Table 2 above, (quoted to three significant figures) without adjustment for the constant factor of 10⁴.

• Calculation of growth rates Concentration-effect relationships were calculated by comparing growth rates in control and test cultures in the following way. The average specific growth rate (u) for individual cultures was calculated from the following relationship:

  u = 1n (Nn) - 1n (N0)

  t_n- t₀ Where 1n (Nn) = natural logarithm of cell number at time tn; 1n (N0) = natural logarithm of cell number at time t0; t₀= nominal measurement at the beginning of the test; t_n= time of nth measurements after the beginning of the test. The average specific growth rate of the test culture was expressed as a percentage of the solvent control, and subtracted from 100 to give the percentage effect. The ErC50 could not be calculated because no inhibition of algal growth occurred.

• Calculation of the areas under the growth curves Biomass was calculated by determining the areas under the growth curves of control and test cultures in the following way:

A = N1– N0    X    t1       +     N1+ N2– 2N0       X     (t2–t1)

              2                                       2                      

 

                +    Na-1+ Nn– 2N0             X            (tn–tn-1)

                              2

 

Where:

A = area;

N0= nominal number of cells/ml at t0;

N1= measured number of cells at t1;

N2= measured number of cells at t2;

Nn = measured number of cells at timetn;

T1= time of first measurement after start of test;

t2= time of second measurement after start of test;

tn= time of n^thmeasurement after start of test.

The area under the growth curve (biomass) at the test concentration was expressed as a percentage of the solvent control and subtracted from 100 to give the percentage inhibition. The EC50 based on biomass (EbC50) could not be calculated because insufficient inhibition of algal growth was noted. 

A statistical comparison of the area under the growth curve in the solvent control and test groups was again carried out using Dunnett’s multicomparison test.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Selenastrum capricornutum to an aqueous mixture of test item, derived from a preparation at an initial nominal level of100 mg/Litre, did not result in a significant reduction of either the average specific growth rates or the mean biomass values compared to control cultures (p >0.05). Neither the median effect concentration (ErC50 or EbC50) nor the no-observed-effect concentrations could be identified but both must be at least equal to the limit of aqueous solubility of the test material.

Executive summary:

A study was conducted to investigate the effects of test item on the growth of the unicellular green alga Selenastrum capricornutum over a 72 hour period. The study was conducted in mineral salts medium at temperatures in the range 21.6 to 25.3 °C in an illuminated orbital incubator using methods based on OECD Procedure 201 and the Annex to EC Directive 92/69/EEC, Part C3. The exposure concentration (100mg/Litre) was not verified during the limit test because attempts to develop an appropriate method of chemical analysis were unsuccessful. The formulation methods employed are considered to have given rise to the maximum attainable exposure concentration of test item under the conditions of the test. Exposure of Selenastrum capricornutum to an aqueous mixture of the test substance did not result in a significant reduction of either the average specific growth rates or the mean biomass values compared to solvent controls (p >0.05). Neither the median effect concentration (ErC50 or EbC50) nor the no-observed-effect concentrations could be identified but both must be at least equal to the limit of aqueous solubility of the test material.

Description of key information

A study was conducted to investigate the effects of test item on the growth of the unicellular green algaSelenastrum capricornutumover a 72 hour period using methods based on OECD Procedure 201 and the Annex to EC Directive 92/69/EEC, Part C3. Exposure of Selenastrum capricornutumto an aqueous mixture of the test substance did not result in a significant reduction of either the average specific growth rates or the mean biomass values compared to solvent controls (p>0.05). Neither the median effect concentration (ErC50 or EbC50) nor the no-observed-effect concentrations could be identified but both must be at least equal to the limit of aqueous solubility of the test material.

Additional information

Neither the median effect concentration (ErC50 or EbC50) nor the no-observed-effect concentrations could be identified but both must be at least equal to the limit of aqueous solubility of the test material.