4,4'-methylenebis[N-sec-butylaniline]

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:

08 Jun 2018 - 27 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006; Annex 5 corrected 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of three hours. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- 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 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- Method of cultivation: 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 21-24°C.
- 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 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1 medium, according to NPR 6505
- Pre-culture medium and test medium: M2 medium, according to OECD 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22 - 23°C

pH:

At t=0 h: 8.2
At t=72 h: 8.1 - 8.3

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L
Measured concentrations: 0.000096, 0.00098, 0.022, 0.13, 0.50 mg/L (Time Weighted Average concentrations, see 'Any other information on results' for details on measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 145 x 10^4 cells/mL
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations and the control. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 96 to 98 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the 10% SS test group and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: 1.0, 10, 100 % of SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jul 2018)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

187 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 183-192 ug/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

81 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 78-85 ug/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.98 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Details on results:

- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 22 µg/L when compared to the control.
- Growth rates were in the range of the control at the two lowest test concentrations, while inhibition of growth rate increased with increasing concentration from 22 µg/L upwards, resulting in 99% inhibition at the highest concentration. Statistically significant inhibition of growth rates was found at the three highest test concentrations. However, growth rate inhibition was considered to be biologically not relevant at 22 µg/L, where the observed inhibition was below 10%.
- Analytical results: The concentrations measured at the start of the test were 0.0060, 0.022, 0.082, 0.25 and 0.76 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations decreased significantly and were below 30% relative to the initial concentrations at the end of the test in the two highest test concentrations. In the three lowest test concentrations, no concentrations could be detected anymore at the end of the test. The concentrations measured in the samples without algae were comparable to the concentrations measured in the samples with algae.
- It should be noted that a concentration of 0.055 mg/L was originally measured in the control at the start of the test, while no concentrations could be detected in the samples of the control taken after 24 and 72 hours of exposure. No reason could be identified for this and it was decided to analyse the corresponding reserve sample to investigate whether the sample was contaminated before or after sampling. The concentration detected in the reserve sample was at the level of the carry-over effect observed during the analysis of the samples. As such, it was concluded that the control was not contaminated and that the originally determined concentration was caused by contamination during sampling or preparation of the sample for analysis.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 - 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

- Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
- ECx calculation: probit analysis using linear max. likelihood regression.
- For the determination of ECx-values for growth rate inhibition, the test group exposed to the lowest test concentration, was excluded from the model fitting procedure. Including the lowest test group interfered with identifying an appropriate non-linear or linear regression model for the calculation of ECx-values. Since no inhibition of growth rate was observed at the two lowest test concentrations the exclusion of the lowest test group was considered to not have impacted the determined ECx-values.
- ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Time Weighted Average Concentrations

4,4’-methylenebis-N-sec-butylaniline %SS prep. at 100 mg/L	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.00604	0.000012 (b)	0.000012 (b)	0.000096
3.2	0.0224	0.00035	0.000012 (b)	0.00098
10	0.0817	0.0513	0.000012 (b)	0.022
10 (a)	0.0777	0.0294	0.000012 (b)	0.016
32	0.248	0.197	0.041 (c)	0.13
100	0.762	0.710	0.206	0.50

(a) Without algae.; (b) Estimated as half of the LOD (LOD = 0.023 µg/L); (c) Average of two analyses with different dilutions of the sample.

 

Table 3: Growth Rate and Percentage Inhibition for the Total Test Period

4,4’-methylenebis-N-sec-butylaniline TWA conc. (µg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.659	0.0227	6
0.096	1.663	0.0318	3	-0.26
0.98	1.697	0.0127	3	-2.3
22	1.504	0.0361	3	9.4^#
134	1.231	0.0408	3	26 *
500	0.009	0.0148	3	99 *

* Effect was statistically significant.;^#Effect was statistically significant but biologically not relevant (<10%).

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L (95% confidence interval: 183 - 192 µg/L), based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance.

Executive summary:

In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to saturated solutions of the test substance prepared at a loading rate of 100 mg/L (1.0, 3.2, 10, 32, 100 % of SS, 3 replicates each) and an untreated control (6 replicates). Measured concentrations of test substance decreased during the exposure period, therefore Time Weighted Average concentrations were calculated and effect parameters were based on TWA concentrations. The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L with a 95% confidence interval ranging from 183 to 192 µg/L, based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC for growth rate inhibition was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.

Description of key information

72h-ErC50 (growth rate): 187 ug/L (time weighted average concentration)

72h-ErC10 (growth rate): 81 ug/L (time weighted average concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

187 µg/L

EC10 or NOEC for freshwater algae:

81 µg/L

Additional information

In a 72h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to saturated solutions of the test substance prepared at a loading rate of 100 mg/L (1.0, 3.2, 10, 32, 100 % of SS, 3 replicates each) and an untreated control (6 replicates). Measured concentrations of test substance decreased during the exposure period, therefore Time Weighted Average concentrations were calculated and effect parameters were based on TWA concentrations. The 72h-EC50 for growth rate inhibition (ErC50) was 187 µg/L with a 95% confidence interval ranging from 183 to 192 µg/L, based on Time Weighted Average concentrations. The 72h-ErC10 was 81 µg/L (95% confidence interval: 78 - 85 µg/L). The 72h-NOEC for growth rate inhibition was 0.98 µg/L based on statistical significance and 22 µg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.