Ammonium bromide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Ammonium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to ammonium bromide.

Analytical monitoring:

no

Details on sampling:

None stated

Vehicle:

no

Details on preparation and application of test substrate:

Type and source of dilution water - Deionised reverse osmosis water prepared in house.
Vehicle control performed - Yes, Water only added to artificial soil and maintained under the same conditions as the test vessels

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Criteria Details
Species/strain Eisenia foetida
Source of the initial stock Blades Biological, Cowden, Edenbridge, Kent, UK
Culturing techniques Maintained in 20 L plastic trays at 22°C on a substrate consisting of 50% by volume peat and 50% by volume animal manure.
Age/weight At least 2 months old with clitellum
Mean weight: 0.4 g
Pre-treatment Transferred to artificial soil without test material 24°h before start of test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

21C to 23ºC

pH:

6.0 ± 0.5

Moisture:

Experience has shown that a moisture content of 35% of dry weight, as stated in the Test Guideline, may be too high for the preparation of the artificial soil, causing sludge formation. Therefore, a moisture content of 30% of dry weight was considered appropriate for use during the study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive test was 28% to 29% of dry weight of basic substrate on Day 0 and 26% to 27% on Day 14.

Details on test conditions:

Criteria Details
Artificial soil test substrate Industrial quartz sand: 70% w/w
Kaolinite clay: 20% w/w
Sphagnum moss peat:10% w/w
Test mixture 50 mL stock solution :05 kg dry weight soil + 90 mL water
Size, volume and material of test container 1L glass beaker
Amount of artificial soil (kg)/ container 0.5 kg dry weight
Nominal levels of test concentrations Range-finding study: 10, 100 and 1000 mg bromide/kg
Initial study: 1000 mg bromide/kg
Definitive study: 10, 32, 100, 320 and 1000 mg bromide/kg
Number of replicates/concentration 1 per concentration
Number of earthworms/test concentration 10 per concentration
Number of earthworms/container 10
Light source Not specified
Test performed in closed vessels due to significant volatility of test substrate No

Nominal and measured concentrations:

0, 10, 32, 100, 320, 1000 mg/kg

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/kg soil dw

Nominal / measured:

nominal

Basis for effect:

other: bodyweight

Results with reference substance (positive control):

. A positive control using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the probit method (Finney 1971) on Day 7 and by the trimmed Spearman-Karber method (Hamilton et al 1977) on Day 14 using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate
Statistical analysis of the earthworm weight data was performed using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnet 1995). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001)

Validity criteria fulfilled:

yes

Conclusions:

A study to OECD 207 was performed to assess the acute toxicity of sodium bromide to the earthworm (Eisenia foetida) in an artificial soil.
Following a preliminary range-finding test and an initial experiment, earthworms were exposed in groups of 40 (four replicates of 10 worms per concentration) to a range of concentrations of 10, 32, 100, 320 and 1000 mg bromide/kg of soil for a period of 14 days at a temperature of 21ºC to 24°C. The number of mortalities were determined after 7 and 14 days exposure. A positive control using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes
The No Observed Effect Concentration (NOEC) was 10 mg bromide/kg on the basis that no mortalities were observed after 14 days exposure and additionally no sub-lethal effects on weight or behaviour were observed at 10 mg bromide/kg.
The result of the positive control gave a 14-Day LC50 for chloroacetamide of 24 mg/kg with 95% confidence limits of 23 – 25 mg/kg. The No Observed Effect Concentration was 18 mg/kg.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):

Ammonium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to ammonium bromide.

Analytical monitoring:

yes

Details on sampling:

NaBr (9.0175mg of 99.92% analytical standard) dissolved in water (9.010 mL) in an ultrasonic bath for 5 mins to obtain a fortified solution of 1000 μg/mL.
Calibration solution range: 0.1 – 10 μg/mL (stored at 5°C)
Untreated soil fortified with 1 mg/kg and 100 mg/kg NaBr.
10g dry soil weighed into 50 mL centrifuge tube. Untreated soil fortified at this stage. 40 mL water added (minus amount from artificial soil) and shaken for 30 mins, then centrifuged for 10 mins at 4000 rpm. An aliquot was filtered through a 0.45 μm PTFE filter into a 2 mL HPLC vial.
Soil samples were taken directly after application and 56 days after application for analysis of the test item in soil - samples were not tored prior to analysis.

Vehicle:

no

Details on preparation and application of test substrate:

The test item was applied to the artificial soil by means of an aqueous application solution.
Control: Yes, negative control , no test item and a positive control with Derosal

Test organisms (species):

Eisenia fetida

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: synchronized culture maintained at Harlan Laboratories
- Age at test initiation (mean and range, SD): 3–4 months old adults with a clitellum
- Weight at test initiation (mean and range, SD): 304 to 598 mg

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): None stated.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

adults and juveniles exposed for 4 weeks each.

Post exposure observation period:

None

Test temperature:

19-21°C

pH:

5.9 - 6.3

Moisture:

40%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm2)
- Amount of soil or substrate: 510 g of the prepared artificial soil (corresponding to 500 g dry weight) was mixed with 2.5 g calcium carbonate and 5 g food in a laboratory mixer. During mixing, 100 mL of purified water, containing the desired concentration of the test item was added. Finally, 90 mL of purified water was added.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4 for positie control, 8 for negative control


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
The artificial soil was prepared according to the guidelines with the following constituents
(percentage distribution on dry weight basis):
- Sphagnum peat: 10%
(shredded, sieved through 2 mm sieve)
- Kaolinite clay: 20%
content of Al2O3: 35.1%
content of Kaolinite: approx. 85%
- Sand (Sihelco 36): 69%
Size analysis:
0.25 mm 1%
0.18 mm 8%
0.125 mm 80%
0.09 mm 10%
<0.09 mm 1%
- Calcium carbonate (CaCO3): about 0.5%
(for the adjustment to pH 6.0 ± 0.5)
- Food (dried organic manure): 1%

- Maximum water holding capacity (in % dry weigth): 60%
- Stability and homogeneity of test material in the medium: stable for 56 days (see analytical section)

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness photoperiod
- Light intensity: 506-670 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After four weeks of exposure, the content of each test vessel was emptied and the living adult worms were counted and checked for any abnormal behavior or other adverse effects (worms not moving after gentle mechanical stimulus to their front end are considered to be dead, also missing worms are considered to have died since dead worms can completely decompose in the soil in short time and hence cannot always be found).
At the beginning (prior to exposure) and end of the first four weeks of the test, the adult test organisms of each test vessel were weighed (at the start each individually, at the end all together in each test vessel). Before weighing, the worms were quickly washed with water and surplus water was adsorbed on filter paper.
For each test vessel, the difference of the mean body wet weight of the surviving test organisms between start and end of the exposure period was calculated. The changes in mean body weight of the surviving worms were compared to the control, and were statistically evaluated by means of a multiple Dunnett-t test after a one-way analysis of variance (ANOVA).
The body weight change of the surviving worms in the positive control was compared to the control and was statistically evaluated by means of a Student-t test, α = 0.05, one-sided smaller

At the test termination after 8 weeks, the number of living juveniles per test vessel was determined. The test vessels were warmed up in a water bath at 60 °C for approximately 15-20 minutes to encourage the living juvenile earthworms to rise to the soil surface. The juvenile earthworms at the soil surface were counted and removed. The test vessels were warmed up for a second time for about 10 minutes and the surface was searched for juveniles. Afterwards, each test vessel was emptied and the soil was intensely searched for eventually remaining juveniles.
The reproduction rates of the surviving test organisms at the end of the test were compared to the control values and were statistically evaluated by means of a multiple Dunnett-t test after a one-way analysis of variance (ANOVA).

At each feeding date, the amount of food consumed by the adult worms was visually estimated for each test vessel

The water holding capacity (WHC) was determined in two samples of the artificial soil and the moisture of the test substrate was adjusted . At the start of the test, the soil moisture was checked in samples from the treatments.

At the start of the test, the pH of the untreated artificial soil was determined in representative samples. At the end of the test, the pH of the soil was checked again for all treatment groups and the control.

The test temperature (room temperature) was continuously monitored by a digital temperature recorder

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study, no, acute toxicity study with earthworm available.

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg per kg dry soil.

Reference substance (positive control):

yes

Remarks:

Derosal (8.33 mg/kg dwt)

Duration:

4 wk

Dose descriptor:

LC50

Effect conc.:

92.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: no CI determined due to a steep concentration-effect relationship

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

53 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI = 38 – 75 mg/kg dwt

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

32 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
The mean body weight of the adult worms in the control had decreased during the 4 weeks exposure period in average by 20 mg per worm (5% of the mean initial weight).
The mean decrease in body weight was statistically significantly higher compared to the control first at the highest nominal test concentration of 100 mg/kg dry soil (results of a Dunnett-t test, one-sided smaller, α = 0.05). Moreover, the food consumption at the highest test concentration was found to be slightly lower when compared to the control and to the other treatment groups.

- Reproduction
In the control, an average of 84 juvenile worms per test vessel were found (corresponding to a mean reproduction rate of 8.4 juveniles per surviving adult). The coefficient of variance of the reproduction rate per test vessel and per surviving individual in the control was 14%. Thus, the validity criteria of the test guidelines (at least 30 juveniles per test vessel, coefficient of variance of reproduction ≤30%) were well fulfilled.
According to the results of a Dunnett-t test (one-sided smaller, α = 0.05), the mean reproduction rate of the worms was statistically significantly reduced first at the highest nominal test item concentration of 100 mg/kg dry soil, where this value was 15% of the control.

Results with reference substance (positive control):

At the single concentration of the reference item Derosal® (8.33 mg/kg formulation = 5 mg a.i./kg), the mortality rate of the adult worms was 5%
The mean decrease of 30% in body wet weight of the earthworms in the positive control during the exposure period was statistically significantly different to the control (results of a Student-t test, one-sided smaller, α = 0.05).
The reproduction rate of Eisenia fetida in the positive control was completely inhibited

See results tables in attached document.

Validity criteria fulfilled:

yes

Conclusions:

The highest concentration of Sodium Bromide (NaBr) without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be 32 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) was 100 mg/kg dry soil due to a high mortality, statistically significant change in mean body weight, reduced food consumption and reduced reproduction rate of the worms at this test concentration

Executive summary:

Materials and method

The toxic effects of Sodium Bromide (NaBr) on survival, growth, and reproduction of the

earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guideline 222.

Sodium Bromide (NaBr) was homogeneously mixed into an artificial soil at the following test concentrations: 1.0, 3.2, 10, 32 and 100 mg per kg dry soil. Additionally, a control and a reference item treatment were tested in parallel. Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Results and discussion

In the control group and at the nominal test concentrations up to and including 32 mg/kg dry soil, no mortality of the adult test organisms was observed. At the highest test item

concentration of 100 mg/kg dry soil, the mortality rate was 58%.

The mean body weight of the adult worms in the control had decreased during the 4 weeks

exposure period in average by 20 mg per worm (5% of the mean initial weight). Moreover, the food consumption at the highest test concentration was found to be slightly lower when compared to the control and to the other treatment groups.

In the control, an average of 84 juvenile worms per test vessel were found (corresponding to a mean reproduction rate of 8.4 juveniles per surviving adult). The mean reproduction rate of the worms was statistically significantly reduced first at the highest nominal test item concentration of 100 mg/kg dry soil, where this value was 15% of the control.

Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of Sodium Bromide (NaBr) without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be 32 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) was 100 mg/kg dry soil due to a high mortality, statistically significant change in mean body weight, reduced food consumption and reduced reproduction rate of the worms at this test concentration.

The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions

Description of key information

The toxic effects of Sodium Bromide (NaBr) on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guideline 222. Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of Sodium Bromide (NaBr) without toxic effects was determined to be 32 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) was 100 mg/kg dry soil due to a high mortality, statistically significant change in mean body weight, reduced food consumption and reduced reproduction rate of the worms at this test concentration.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

32 mg/kg soil dw

Additional information

Ammonium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to ammonium bromide.

Acute

A study to OECD 207 was performed to assess the acute toxicity of sodium bromide to the earthworm (Eisenia foetida) in an artificial soil. Earthworms were exposed to a range of concentrations of 10, 32, 100, 320 and 1000 mg bromide/kg of soil for a period of 14 days. The number of mortalities were determined after 7 and 14 days exposure. The No Observed Effect Concentration (NOEC) was 10 mg bromide/kg on the basis that no mortalities were observed after 14 days exposure and additionally no sub-lethal effects on weight or behaviour were observed at 10 mg bromide/kg.

Long term

The toxic effects of Sodium Bromide (NaBr) on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guideline 222.

Sodium Bromide (NaBr) was homogeneously mixed into an artificial soil at the following test concentrations: 1.0, 3.2, 10, 32 and 100 mg per kg dry soil. Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of Sodium Bromide (NaBr) without toxic effects was determined to be 32 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) was 100 mg/kg dry soil due to a high mortality, statistically significant change in mean body weight, reduced food consumption and reduced reproduction rate of the worms at this test concentration.