Bronopol

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

2000

GLP compliance:

yes

Analytical monitoring:

not specified

Remarks:

Gas chromatography indicated for both nitrapyrin and dinoterb. The guidelines do not require analytical confirmation of the test item of nominal concentrations in the soil samples.

Details on sampling:

Endpoints were determined for all treatments in both the nitrification and CO2 respiration tests at the intervals of 0 days (less than 3 hours) and 28 days after treatment.

Vehicle:

no

Details on preparation and application of test substrate:

A geometric series of five concentrations of Bronopol was tested. Soil samples were treated with the test item at rates corresponding to concentrations of 10 mg (treatment I), 33 mg (II), 111 mg (III), 333 mg (IV) and 1000 (V) mg Bronopol per kg dry soil. Test material was applied to the soil using a quartz sand carrier. Soil samples of approximately 150 g dry weight were set-up in 1-litre incubation flasks closed with cotton wool plugs and incubated in the dark at 20 ± 2°C.

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

In this study, one fresh agricultural sandy loam soil, was moistened to 45% of its maximum water-holding capacity and incubated in the dark at 20 ± 2°C following treatment with the test item.

Details on test conditions:

The incubation temperature was monitored continuously. The moisture content of the samples was monitored on a weekly basis and moisture loss was compensated by the addition of purified water. Thereafter, the soil samples were thoroughly mixed.

Nominal and measured concentrations:

Nominal concentrations: 10, 33, 111, 333 and 1000 mg/kg

Reference substance (positive control):

yes

Remarks:

Nitrification Test: Nitrapyrin (0.005 mg/g dry weight soil) Respiration Test: Dinoterb (0.025 mg/g dry weight soil)

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

11.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

11.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

78.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

10.4 mg/kg soil dw

95% CI:

>= 2.41 - <= 21.8

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

31.2 mg/kg soil dw

95% CI:

>= 12.6 - <= 52.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

104.4 mg/kg soil dw

95% CI:

>= 64.87 - <= 166.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Details on results:

Nitrification Test:
For the nitrification part, in the control samples of soil amended with lucerne meal, the mean nitrite concentration on day 0 was 0.20 mg/kg dry soil. On day 28, no nitrite could be detected in the control.
For treatments I to V, the mean initial concentrations of nitrite were 0.12 mg NO2-/kg dry soil (treatment I), 0.12 mg/kg (II), 0.19 mg/kg (III), 0.32 mg/kg (IV) and 0.61 mg/kg (V), respectively. On day 28, no nitrite was detected for treatments I, II and III. For treatments IV and V, 1.16 mg/kg and 21.21 mg/kg were observed.
On day 28 nitrate amounted to 91.5 mg/kg dry soil in the control sample amended with lucerne meal and to 106.7 mg/kg, 110.5 mg/kg, 148.7 mg/kg, 65.0 mg/kg and 102.0 mg/kg for treatments I to V, respectively. The calculated deviations from the control were 16.6%, 20.8%, 62.5%, -28.9% and 11.5%, respectively.
In conclusion, after 28 days, a dose-dependent influence of Bronopol on the nitrate concentration in lucerne amended soil was observed for concentrations 10 mg/kg to 111 mg/kg. At the two highest Bronopol doses (333 mg/kg and 1000 mg/kg), a different mechanism of influence on the nitrification process took place.

Respiration Test:
For the control and for the treated samples 3.86 mg (treatment I), 3.23 mg (II), 2.25 mg (III), 0.85 mg (IV) and 0.66 mg CO2/h (V). In comparison to the untreated control, increasing deviations in respiration of -11.1% (treatment I), -25.5% (II), -48.2% (III), -80.4% (IV) and -84.8% (V) were observed for the treated samples.
Thus, no significant influence of the test item on soil microbial respiration was observed at a concentration of 10 mg/kg dry soil. At concentrations of 33 mg test item /kg dry soil or above, respiration is significantly reduced (>25% effect), showing that the microflora was impaired by Bronopol.


For further details please see section: "Any other information on results incl. tables".

Results with reference substance (positive control):

For further details please see section: "Any other information on results incl. tables".

Reported statistics and error estimates:

The Dixon-test, as reported by Sachs (1984) or Dixon (1953) was used to eliminate outliers in the nitrification experiments. The test was performed with three values (n = 3) and a confidence interval of 90%.
Comparison of Mean Values: The mean of individual values of the nitrification experiments at the end of their respective incubation period were statistically evaluated by Dunnett’s t-test (two-tailed, 5%) to find significant differences between control and treated sample.
Determination of Microbial Respiration and Biomass: As outlined under Section 2.5.1.1, the CO2 evolved from each sample was calculated based on the accumulation time, the flow rate, the measurement period, and the concentration of carbon dioxide in the outlet air stream. From these data, the carbon dioxide production rate was calculated. For the calculation of the microbial biomass the initial and constant CO2 production rate per kg of dry soil (VCO2) and Anderson and Domsch equation were used.
Nitrite- or Nitrate- Formation: The concentration of nitrite and nitrate in the soil samples was calculated.
Calculation of ECx values: The respiration rates for treated and control samples of day 28 were used to calculate % inhibition values. These percentages were plotted against concentration and the EC10, EC25 and EC50 for respiration and the 95%-confidence limit were calculated by probit analysis.
Similar, the nitrate concentrations of day 28 were used to calculate % stimulation values. The EC10, EC25 and EC50 for nitrification were calculated as described above.

Table1:  Influence of the test item and nitrapyrin on nitrite formation after amendment of lucerne meal.

 

		Nitrite
	Incubation	[mg NO2^-/ kg dry soil]
	time	Replicates						% Deviation
	[days]	a	b	c	Mean	SD	%SD	from control
Control	0	0.21	0.20	0.20	0.20	0.002	1.2	-------
	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	-------
Treatment I	0	0.1	0.1	0.1	0.12*	0.009	7.4	-40.0
(10 mg/kg)	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
Treatment II	0	0.12	0.12	0.11	0.12*	0.009	7.3	-40.0
(33 mg/kg)	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
Treatment III	0	0.19	0.19	0.18	0.19	0.002	1.3	-5.0
(111 mg/kg)	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
Treatment IV	0	0.32	0.29	0.35	0.32*	0.029	9.0	60.0
(333 mg/kg)	28	1.16	1.16	1.17	1.16*	0.005	0.4	n.a.
Treatment V	0	0.59	0.65	0.60	0.61*	0.031	5.1	205.0
1000 mg/kg)	28	20.95	21.36	21.32	21.21*	0.226	1.1	n.a.
Nitrapyrin	0	0.21	0.20	0.21	0.2	0.004	2.0	5.0
	28	0.09	0.09	0.09	0.09	<0.001	<0.1	n.a.

SD: Standard deviation

n.a.: not applicable

*: Value is significantly different from the control (Dunnett-test, two-sided, α = 0.05)

 

Table 2: Influence of the test item and nitrapyrin on nitrate formation after amendment of lucerne meal.

		Nitrate
	Incubation	[mg NO3^-/ kg dry soil]
	time	Replicates						% Deviation
	[days]	a	b	c	Mean	SD	%SD	from control
Control	0	51.7	51.8	49.6	51.0	1.2	2.4	-------
	28	92.0	92.1	90.5	91.5	0.9	1.0	-------
Treatment I	0	51.0	51.4	51.4	51.3	0.3	0.5	0.5
(10 mg/kg)	28	107.5	106.3	106.4	106.7*	0.7	0.6	16.6
Treatment II	0	51.8	51.3	51.3	51.5	0.3	0.6	0.9
(33 mg/kg)	28	110.6	111.9	109.1	110.5*	1.4	1.3	20.8
Treatment III	0	50.9	51.0	51.5	51.1	0.3	0.6	0.2
(111 mg/kg)	28	150.8	147.8	147.5	148.7*	1.8	1.2	62.5
Treatment IV	0	51.8	51.3	51.7	51.6	0.3	0.5	1.1
(333 mg/kg)	28	65.3	65.5	64.2	65.0*	0.7	1.1	-28.9
Treatment V	0	52.5	51.9	52.1	52.2	0.3	0.6	2.2
1000 mg/kg)	28	101.6	102.7	101.7	102.0*	0.6	0.6	11.5
Nitrapyrin	0	52.1	51.4	51.8	51.8	0.3	0.7	1.4
	28	9.0	9.0	9.0	9.0*	0.0	0.1	-90.2

SD: Standard deviation

*: Value is significantly different from the control (Dunnett-test, two-sided, α = 0.05)

 

Table 3: Influence of the test item and dinoterb on glucose-induced short-term respiration of soil Speyer 2.3.

 

		Respiration Rates
	Incubation	[mg CO2/h per kg dry soil]
	time	Replicates						% Deviation
	[days]	a	b	c	Mean	SD	%SD	from control
Control	0	5.34	5.19	5.17	5.23	0.09	1.8	-------
	28	4.41	4.11	4.51	4.34	0.21	4.9	-------
Treatment I	0	4.19**	2.75	2.30	2.52*	0.32	12.5	-51.8
(10 mg/kg)	28	3.85	4.05	3.69	3.86	0.18	4.7	-11.1
Treatment II	0	1.79	1.21	1.22	1.40*	0.33	23.7	-73.2
(33 mg/kg)	28	3.29	3.58	2.82	3.23*	0.38	11.8	-25.5
Treatment III	0	1.04	1.11	1.19	1.12*	0.07	6.5	-78.7
(111 mg/kg)	28	2.44	2.25	2.06	2.25*	0.19	8.4	-48.2
Treatment IV	0	0.94	0.89	0.88	0.90*	0.04	3.9	-82.8
(333 mg/kg)	28	0.92	0.84	0.79	0.85*	0.07	7.9	-80.4
Treatment V	0	0.79	0.73	0.70	0.74*	0.05	6.1	-85.3
1000 mg/kg)	28	0.69	0.67	0.62	0.66*	0.04	5.3	-84.8
Dinoterb	0	1.66	2.35	2.39	2.13*	0.41	19.3	-57.7
	28	0.97	0.90	0.88	0.92*	0.05	5.4	-78.9

SD: Standard deviation

*: Value is significantly different from the control (Dunnett-test, two-sided, α = 0.05)

**: Outlier; not used for the calculation of the mean

 

Validity criteria fulfilled:

yes

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

10.4 mg/kg soil dw

Additional information

The adverse effects of Bronopol on aerobic soil microorganisms were investigated by means of the nitrate production test according to OECD guideline 216 with Lucerne meal as nitrogen source. The content of nitrate in aqueous soil extracts was determined by a colometric method. The concentration is measured colorimetrically at a wavelength of 550 nm. The percentage of inhibition of the nitrate production in the treated soil samples versus untreated controls was calculated.The EC10 on nitrogen transformation after 28 days exposure to Bronopol was 11.5 mg/kg dry matter soil; the corresponding EC50 after 28 days = 78.1 mg/kg dry matter soil.

A second study for the same purpose has been conducted by means of a carbon transformation test according to OECD guideline 217, which measures the carbon transformation by the glucose induced soil respiration. The respiration rates were measured for 18 consecutive hours by infrared gas analyzer that measured the evolved CO2. The EC10 on carbon transformation after 28 days exposure to Bronopol was determined to be 10.4 mg/kg dry matter soil; the corresponding EC50 after 28 days was 104.4 mg/kg dry matter soil.