N-bromosuccinimide

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:

03 March 2014 to 14 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 36.5%

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

yes

Remarks:

1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 36.5%

Test guideline 4

Qualifier:

according to guideline

Guideline:

other:

Deviations:

no

Remarks:

Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Principles of method if other than guideline:

-

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-bromosuccinimide

EC Number:

204-877-2

EC Name:

N-bromosuccinimide

Cas Number:

128-08-5

Molecular formula:

C4H4BrNO2

IUPAC Name:

1-bromopyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: N-Bromo-succinimide
Molecular formula: C4H4BrNO2
Molecular weight: 177.98
CAS Number: 128-08-5
Description: Slightly yellowish powder with lumps (determined at WIL Research Europe B.V.)
Batch: LSNB5A8031
Purity/Composition: 99.4%
Test substance storage: In refrigerator (2-8°C) under nitrogen protected from light
Stable under storage conditions until: 31 December 2014 (expiry date)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Volatile: No
Reactivity: Reactive to light and moisture
Stability in water: No
Solubility in water: 14.8 g/L at 20°C
Test substance handling: Use amber-coloured glassware or wrap container in
aluminium-foil. Flush container with nitrogen after handling

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 0.4 ml
Storage: Not applicable, samples were analysed on the day of sampling.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 0.4 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of N-Bromo-succinimide tested was a slightly yellowish powder with lumps with a purity of 99.4% and completely soluble in test medium at the concentrations tested.

Preparation started with a stock solution, i.e. 100 mg/l applying a 10-minute treatment period with ultrasonic waves to accelerate the dissolving of the test substance in the test medium. Thereafter, the obtained clear and slightly yellow solution was magnetically stirred for a period of 2 hours to assure conversion to the degradation product, i.e. Succinimide.

The pH of the stock was set to 6.4 with 1N NaOH , whereas the pH of the M2 medium was set to 6.7 (range-finding test) and 6.6 (final test) with 1N HCl.

The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless, except for the highest concentration that was slightly yellow.

Because test substance was sensitive to light, all handlings were performed under dimmed light and the glassware was wrapped in aluminium foil.

Test organisms

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 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 10e4 cells/ml.
- 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 (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test conditions

Hardness:

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

Test temperature:

22-23 C

pH:

7.0-7.8

Dissolved oxygen:

not measured

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations: 0.46, 1.0, 2.2, 4.6 and 10 mg/l. Time Weight Average concentrations: 0.40, 0.86, 1.9, 4.0 and 8.6 mg/l, respectively

Details on test conditions:

- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 233 x 10e4 cells/ml
Replicates:
3 replicates of each test concentration.
6 replicates of the control.
1 replicate of each concentration without algae.
1 or 2 replicates of each test concentration for sampling purposes after 24 hours of exposure.


GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 88 to 93 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC10, 20,50, 72 h EyC10, 20, 50

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.2-1.6

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.7-2.1

Effect concentrations 3

Duration:

71 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3.5-3.9

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.34-0.52

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.53-0.73

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other:

Remarks on result:

other: 1.2-1.5

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 1.7 mg/l with a 95% confidence interval ranging from 1.6 to 1.8 mg/l.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Note that NOEC for growth inhibition could not be determined due to mathematical reasons, instead EC10 values was given.

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions GmbH, Germany).

Any other information on results incl. tables

Group mean percentage inhibition of growth rate (total test period)

N-Bromo-succinimide TWA concentration (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.816	0.0263	6	-
0.40	1.771	0.0275	3	2.5^#
0.86	1.700	0.0323	3	6.4^#
1.9	1.474	0.0188	3	19
4.0	0.852	0.1165	3	53
8.6	0.217	0.0116	3	88

^#- effect was considered biologically insignificant (i.e. <10%)

 

Group mean percentage inhibition of growth rate at different time intervals

N-Bromo-succinimide TWA concentration (mg/l)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.6	0.0	1.5	0.0	1.3	0.0
0.40	3	2.3	9.1	1.6	-3.5	1.4	-3.4
0.86	3	2.2	14.0	1.5	1.6	1.4	-2.6
1.9	3	1.7	32.1	1.2	21.8	1.5	-9.9
4.0	3	1.2	53.5	0.3	83.0	1.1	18.6
8.6	3	1.0	59.6	-0.2	114.9	-0.2	112.1

 

Group mean percentage inhibition of yield

N-Bromo-succinimide TWA concentration (mg/l)	Mean	Std. Dev.	n	% Inhibition
Control	232	18.71	6	-
0.40	202	16.89	3	13
0.86	163	15.62	3	30
1.9	82	4.67	3	65
4.0	12	4.65	3	95
8.6	0.92	0.07	3	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, the EC50 for growth rate inhibition (72h-ERC50) was 3.7 mg/l with a 95% confidence interval ranging from 3.5 to 3.9 mg/l based on a TWA concentrations.

The EC50 for yield inhibition (72h-EYC50) was 1.3 mg/l with a 95% confidence interval ranging from 1.2 to 1.5 mg/l based on a TWA concentrations.

The 72h-NOEC for both growth rate and yield inhibition could not be determined.

The EC10 for growth rate inhibition (72h-ERC10) was 1.4 mg/l with a 95% confidence interval between 1.2 and 1.6 mg/l.

The EC10 for yield inhibition (72h-EYC10) was 0.43 mg/l with a 95% confidence interval between 0.34 and 0.52 mg/l.

Executive summary:

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 2012 and the OECD series on testing and assessment number 23, 2000.

The batch of N-Bromo-succinimide tested was a slightly yellowish powder with lumps with a purity of 99.4% and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominally 0.46, 1.0, 2.2, 4.6 and 10 mg N-Bromo-succinimide per litre. Initial cell density was 104 cells/ml. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

The actual concentrations in the two highest groups were at the level of 75-77% of nominal and remained fairly stable during the test (100-125% of initial). In the lower groups, the actual concentrations were below the lowest analytical standard from the start of the test, i.e. 2.4 mg/l.

Because the test substance was considered pure and the concentrations were prepared as dilutions of the highest concentration, it was decided to calculated a TWA concentration for the nominal concentration of 10 mg/l (TWA = 8.6 mg/l) and calculate the remaining concentrations based on the dilution factor of 2.2. The calculated TWA concentrations corresponded to 0.40, 0.86 and 1.9 mg/l for nominally 0.46, 1.0, 2.2 and 4.6 mg/l, respectively.

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