Prometryn

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

Study period:

15 Apr 1985 to 30 Jul 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

not compliant GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two agricultural soils, a loamy sand and a sandy loam, were incubated with the test chemical under various conditions. The test did not follow established test guidelines nor GLP. The test substance concentration in the soils was 5 and 50 mg/L of soil. These figures are corresponding a field application rate of 2.5 kg a.i./ha and the ten-fold rate. The respiration test was conducted for 28 days and the nitrification test was performed for 42 days.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

SOIL RESPIRATION
The treatment of the soils was based on the maximum recommended field rate of the test substance (2.5 kg a. i./ha). Assuming a uniform distribution of the test article in the top 5 cm of the soil, the above mentioned recommended field rate corresponds to 5 mg/L of soil, ten times this rate corresponds to 50 mg/L of soil.
Based on the wet volume to dry weight ratio, the following amount of the test substance were added as acetone stock solutions to the soil corresponding to 200 g of dry weight:

Soil I (Collombey): 0.966 mg/200 g (193.2 ml of wet soil) and 9.66 mg/200 g (193.2 ml of wet soil)
Soil II (Strassenacker): 1.172 mg/200 g (234.4 ml of wet soil) and 11.72 mg/200 g (234.4 ml of wet soil)

The total quantity of acetone in the soil amounted to 0.5% (v/wt). The acetone solution was applied to the surface of the soil. After evaporation of the solvent, the soil was thoroughly mixed. In addition to the test substance treated soil samples, the corresponding untreated soil samples were incubated. The amount of acetone in the control samples was the same as in the test substance treated samples.

SOIL NITRIFICATION
The test substance treatments were performed in the same way as the soil respiration.

Test organisms (inoculum):

soil

Total exposure duration:

42 d

Remarks:

For the respiration test, the exposure duration was 28 days.

Test temperature:

20 ± 1 °C

Moisture:

- Soil Respiration: The soil moisture content of the two soils (I: Collombey; II: Strassenacker) was established at 40% of the maximum water holding capacity (MWC): Soil I: 14.36% (based on the soil dry weight) and Soil II: 17.48% (based on the soil dry weight)
- Soil Nitrification test: The soil moisture content of both soils was adjusted to 40% of their MWC

Organic carbon content (% dry weight):

0.63

Details on test conditions:

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Two field soils were used. They were Collombey (was classified as a loamy sand) and Strassenacker (was classified as a sandy loam). Specifications of the soil are provided in Table 1 in "Any other information on materials and methods incl. tables".

TEST SYSTEM FOR SOIL RESPERATION
- Test container: All glass respiration flasks (in dark)
- Amount of soil: 200 g soil (dry weight) per flask
- No. of replicates per concentration: 3
- No. of replicates per control: 3

EXPERIMENTAL CONDITIONS FOR SOIL RESPIRATION
- Validation: Continuous; CO2 - free air at a rate of 60 mL/min (the effluent air was passed through a CO2 - trapping system of 50 mL 2N sodium hydroxide solution). The sodium hydroxide solution was renewed after the intervals given under sampling and the CO2-concentration determined.
- Sampling: The amount of CO2 liberated from the soil was determined after 1 , 3 , 7. 14 , 21 and 28 days
- Amendments: 1) Untreated soils without amendment; 2) Treated soils without amendment; 3) Untreated soils mixed with 0.5% lucerne meal on a dry weight basis before adjustment of the moisture content; 4) the test substance treated soils amended.

TEST SYSTEM FOR SOIL NITRIFICATION
- Test container: 250 mL Erlenmeyer flasks closed with cotton - wool plugs (in dark)
- Amount of soil: 100 g soil (dry weight) per flask amended with ammonium sulfate
- No. of replicates per concentration: 3
- No. of replicates per control: 3

EXPERIMENTAL CONDITIONS FOR SOIL NITRIFICATION
- Sampling : Samples were analysed after 0 , 3, 7 daysand after 2, 4 and 6 weeks of incubation
- The soil samples (treated and untreated) were amended with 47.1 mg ammonium sulfate per 100 g dry soil dissolved in the water needed to moisten the soil. This quantity of ammonium sulfate corresponded to 100 ppm of nitrogen.

SOIL RESPIRATION AND DETERMINATION OF CO2
Soil respiration can be regarded as a measure for the general turn-over of organic matter in soil. It is determined by following the evolution of CO2 from the soil samples resulting from microbial activity in the soil. The carbon dioxide evolved from the soil samples was trapped in 2N sodium hydroxide solution. Its concentration was measured directly by means of a Technicon Auto Analyser II equipped with a data processing system. In this automated procedure for CO2 analysis, the sample is aspirated into an acidic diluent stream segmented with CO2 free air. The CO2 is released into the air phase, collected by an air trap, and diffused into a weakly alkaline buffered stream of phenolphthalein. The absorbed CO2 causes a decrease in pH, resulting in a decrease in color intensity proportional to CO2 concentration. The absorbance is measured at 550 nm. The operating concentration range was 40 - 2000 ppm. The lower limit corresponded to a limit of quantitation of 1.0 mg CO2 evolved per soil sample and sampling interval.

EXTRACTION OF AMMONIUM, NITRITE AND NITRATE
Two soil samples each corresponding to 40 g of dry soil were extracted with 40 mL 2N KCl by agitation for 15 minutes. Thereafter, the soil was centrifuged at 3000 RPM for 5 minutes, the supernatant decanted and the extraction repeated. The combined aqueous extracts were adjusted with 2N KCl to 100 mL, and the concentration of ammonium-, nitrite- and nitrate-nitrogen determined by means of the Auto - Analyser.

ANALYSIS FOR AMMONIUM-NITROGEN
The determination of ammonium-nitrogen is based on a colorimetric method in which an emerald-green color is formed by the reaction of ammonium, sodium salicylate, sodium nitroprusside and sodium hypochlorite in a buffered alkaline medium at a pH of 12.8 – 13.0. The ammonium salicylate complex concentration is measured at 660 nm. Limit of quantitation: 0.1 mg NH4-N per 100 g of soil.

ANALYSIS FOR NITRITE-NITROGEN
In this method the nitrite ion reacts under acidic conditions with sulfanilamide to yield a diazo compound which couples with N-1-naphthylethylene-diamine dihydrochloride to form a soluble dye which is colorimetrically determined at 520 nm.
- Limit of quantitation: 0.01 mg NO2-N per 100 g of soil.

ANALYSIS FOR NITRATE-NITROGEN
The nitrate is reduced to nitrite by hydrazine-copper sulfate. The nitrite is then determined as described above. Since the method determines free nitrite and nitrate simultaneously the nitrite value determined in the proceeding step has to be subtracted if necessary. Limit of quantitation: 0.2 mg NO3-N per 100g of soil.

Nominal and measured concentrations:

- There were two types of soil inclded in this test: Collombey (soil I) and Strassenacker (soil II)
- For both resperation and nitrification tests, the two soils experienced six nominal cooncentrations: 0 (untreated soil without amendment), 0 (untreated soil mixed with 0.5% lucerne meal), 5 and 50 mg a.i./L dry soil (without amendment), and 5 and 50 mg a.i./L dry soil (with 0.5% lucerne meal)

Reference substance (positive control):

no

Key result

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

>= 48.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other:

Remarks:

loamy sand soil

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 48.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

respiration rate

Remarks on result:

other:

Remarks:

loamy sand soil

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 58.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

respiration rate

Remarks on result:

other:

Remarks:

sandy loam soil

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

>= 58.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other:

Remarks:

sandy loam soil

Details on results:

Overview of the results are provided in "Any other information on results incl. tables"

SOIL RESPIRATION / ORGANIC NATTER TURN-OVER
Loamy sand (Collombey, Soil I)
Soil respiration in the untreated unamended soil was low when compared with the lucerne meal amended soil. The total quantity of CO2 evolved within four weeks amounted to 147.2 mg per 200 g of soil. In the untreated, amended soil the corresponding figure was 708.4 mg CO2 per 200 g of soil. Treatment of the unamended soil with the test article resulted in a total CO2-evolution of 151.5 and 180.6 mg for the low and high herbicide concentration in the soil, respectively. The respective figures correspond to 102.9% and 122.7% of the control value. Amendment of the soil with 0.5% lucerne meal resulted in a total CO2-evolution of 708.4, 722.6 and 742.0 mg carbon dioxide for the untreated, the 5 mg/L and 50 mg/L treated soil, respectively. When compared with the control the respiration of the treated samples amounted to 102.0% and 102.7% for the low and high dose level treatment, respectively.

Sandy loam (Strassenacker, Soil II)
The respiration of the untreated and unamended soil was found to be lower than in the amended soil accounting within four weeks for 147.5 and 703.8 mg CO2 per 200 g of soil, respectively. Treatment of the unamended soil with the test substance resulted in a total CO2-evolution of 128.3 and 137.2 mg CO2/200 g soil for the low and high treatment, respectively. When compared with the control these figures corresponded to 87.0% and 93.0%, respectively. In the amended soil, CO2-evolution accounted for 110.7% and 104.1% of that of the control soil samples for the 5 mg/L and 50 mg/L treatments, respectively. In conclusion, it can be stated that the test substance has no significant influence on basic respiration and organic matter turn - over of soil microbes up to the ten - fold of the recommended field rate.

AMMONIFICATION – NITRIFICATION
Loamy sand (Collombey, Soil I)
The table shows that the amount of nitrate-nitrogen found after 28 days of incubation in the unamended had increased by 193.5%. In the corresponding treated soils, on average, 201.6% increase of the NO3-N concentration in the untreated, 5 and 50 mg/L of soil treated samples of 214.7, 229.4 and 270.6%, respectively. The concentration of ammonium and nitrite was very low in all soil samples at the beginning and end of the study. No significant difference between treated and untreated soils was observed in most samples. Only in the lucerne meal amended soil treated with 50 mg/L a higher (55.9%) value was found when compared with the control .

Sandy loam (Strassenacker, Soil II)
When compared with the values at the beginning of the study, the nitrate-nitrogen concentration in the unamended soil after 28 days of incubation was found to be 125.6, 160.5 and 155.8% for the untreated, 5 and 50 mg/L of soil treated samples, respectively. The corresponding figures of the amended soil were 179.5, 127.3 and 154.5%. The ammonium and nitrite concentrations were found to be low. In conclusion no negative influence of the herbicide on the formation of nitrate in the unamended and lucerne meal amended soils was observed.

SOIL NITRIFICATION: AMMONIUM CONVERSION
As described the soils were amended with 47.1 mg ammonium sulfate per 100 g of soil (dry weight basis). This figure corresponded to 9.99 mg NH4-N or to 12.8 mg NH4 per 100 g of soil. The practical values at the beginning were, on average, very similar to the theoretical value representing in the loamy sand 9.9 mg NH4-N/100 g of soil and in the sandy loam 11.6 mg NH4-N/100 g.
The conversion rate of ammonium to nitrate was very similar in both soils, i.e., 94.3 and 99.1% of the initial NH4- N were already degraded after two weeks of incubation in the loamy sand and sandy loam, respectively. No significant influence of the test substance on the ammonium degradation was observed.

SOIL NITRIFICATION: NITRITE FORMATION
Nitrite formation was very low in the untreated and in the 5 mg/L treated soils ranging from < 0.01 to 0.02 mg NO2-N/100 g of soil. Only in the 50 mg/L treated loamy sand and sandy loam slightly higher values were found after two weeks of incubation representing 1.44 and 0.88 mg NO2-N/100 g of soil. However, after 28 days the concentration of NO2-N in both soils was 0.01 mg/100 g .

SOIL NITRIFICATION: NITRATE FORMATION
The final results obtained for nitrate were in good agreement with the values expected from the conversion of the theoretical concentration of ammonium in the soil at the beginning of the study, i.e. from the conversion of 9.99 mg NH4-N an amount of 9.99 mg NO3-N would have been expected. Nitrate formation was very similar in the untreated and 5 mg/L treated soils. Only in the 50 mg/L treated soils slightly lower values of NO3-N were found in week two. However, the amount of nitrate found after 42 days of incubation in the loamy sand accounted for 10.0 , 10.2 and 10.4 mg NO3-N/100 g of soil for the untreated, 5 mg/L and 50 mg/L treated soil samples, respectively. The corresponding figures for the sandy loam were 9.1 , 9.2 and 9.5 mg/100 of soil. Therefore, it can be stated that the test substance no significant influence on the nitrification process in the soil up to a concentration of 50 mg/L of soil.

Table 2. Influence of the test substance on respiration of unamended and lucerne meal amended loamy sand soil (Collombey) (mean values given represent mg CO2 evolved per 200 g dry soil. Mean was calculated from three independent soil samples. Per sample, two determinations were performed).  

Soil type	Collombey
Amendment with Lucerne meal (%)	0			0.5
Treatment (mg/L soil)	0	5	50	0	5	50
Day
0 – 1	12.7 ± 2.6	13.8 ±1.4	13.1 ± 6.3	67.8 ± 21.5	71.3 ± 27.3	99.8 ± 8.8
1 – 3	19.5 ± 1.6	17.0 ± 7.6	17.5 ± 6.9	120.9 ± 6.3	112.5 ± 22.7	74.0 ± 26.8
3 – 7	26.2 ± 8.3	28.0 ± 4.9	29.9 ± 4.3	194.0 ± 8.5	202.3 ± 13.2	239.9 ± 34.4
Subtotal	58.4	58.8	60.5	382.7	386.1	413.7
7 - 14	38.0 ± 11.8	29.1 ± 3.4	45.9 ± 5.8	145.6 ± 6.2	142.4 ± 3.3	179.0 ± 15.0
14 – 21	36.8 ± 6.7	40.5 ± 6.6	44.1 ± 0.8	107.8 ± 3.0	113.2 ± 2.1	88.6 ± 5.7
21 – 28	14.0 ± 8.5	23.1 ± 7.5	30.1 ± 7.2	72.3 ± 11.8	80.9 ± 2.4	60.7 ± 0.4
subtotal	88.8	92.7	120.1	325.7	336.5	328.3
Total	147.2	151.5	180.6	708.4	722.6	742.0
% of control		102.9	122.7		102.0	102.7

Table 3. Influence of the test substance on respiration of unamended and lucerne meal amended sandy loam soil (Strassenacker) (mean values given represent mg CO2 evolved per 200 g dry soil. Mean was calculated from three independent soil samples. Per sample, two determinations were performed).

Soil type	Strassenacker
Amendment with Lucerne meal (%)	0			0.5
Treatment (mg/L soil)	0	5	50	0	5	50
Day
0 – 1	12.3± 1.7	11.4± 3.5	12.6± 0.7	101.9± 8.9	106.0± 1.8	106.1± 12.0
1 – 3	9.0± 4.1	9.2± 3.9	11.8± 9.3	157.6± 6.1	143.6± 7.4	155.5± 12.7
3 – 7	38.6± 16.0	30.7± 13.2	24.2± 22.9	174.2± 5.6	230.4± 28.1	198.8± 10.8
Subtotal	59.9	51.3	48.6	433.7	480.0	460.4
7 - 14	31.4± 4.5	30.9± 7.2	38.6± 6.2	127.3± 24.4	139.9± 21.0	133.0± 5.1
14 – 21	29.0± 0.8	26.0± 9.0	30.1± 1.1	70.2± 9.2	88.8± 7.7	65.4± 5.0
21 – 28	27.2± 2.0	20.1± 6.8	19.9± 8.3	72.6± 22.2	70.1± 6.5	73.9± 19.2
subtotal	87.6	77.0	88.6	270.1	298.8	272.3
Total	147.5	128.3	137.2	703.8	778.8	732.7
% of control		87.0	93.0		110.7	104.1

 

Table 4. Influence of the test substance on the concentration of ammonium, nitrite and nitrate (mg/200 g dry soil) in unamended and Lucerne meal amended soils 

Soil type		Collombey
Amendment with Lucerne meal (%)		0			0.5
Treatment (mg/L soil)		0	5	50	0	5	50
	Expos. Period (days)
NH4 – N *	0	0.2	0.2	0.2	0.8	0.8	0.8
	28	0.6	0.5	0.5	0.6	0.5	0.3
NO2 – N *	0	0.02	0.02	0.02	0.03	0.03	0.03
	28	0.02	0.02	0.02	0.03	0.03	0.02
NO3 – N *	0	3.1	3.1	3.1	3.4	3.4	3.4
	28	6.0	5.8	6.7	7.3	7.8	9.2
NO3 – N (%) **		193.5	187.1	216.1	214.7	229.4	270.6

   

Soil type		Strassenacker
Amendment with Lucerne meal (%)		0			0.5
Treatment (mg/L soil)		0	5	50	0	5	50
	Expos. Period (days)
NH4 – N *	0	0.2	0.2	0.2	0.6	0.6	0.6
	28	0.2	0.3	0.2	0.3	0.3	0.3
NO2 – N *	0	0.02	0.02	0.02	0.02	0.02	0.02
	28	0.02	0.02	0.01	0.01	0.01	0.01
NO3 – N *	0	4.3	4.3	4.3	4.4	4.4	4.4
	28	5.4	6.9	6.7	7.9	5.6	6.8
NO3 – N (%) **		125.6	160.5	155.8	179.5	127.3	154.5

* Expressed as ammonium- nitrite - and nitrate-nitrogen .

** NO3-N concentration at day 28 in % of the corresponding value of day 0 .

 

Table 5. Influence of the test substance on nitrification of ammonium sulfate amended soils (values in mg/100 g of dry soil).

Soil type		Collombey			Strassenacker
Treatment (mg/L soil)		0	5	50	0	5	50
	Expos. Period (days)
NH4 – N *	0	9.9	9.9	9.9	11.6	11.6	11.6
	3	8.9	9.2	9.2	9.2	8.9	9.5
	7	6.9	6.7	7.7	7.8	8.2	8.4
	14	0.1	0.1	1.5	0.1	0.1	0.1
	28	0.2	0.2	0.2	0.1	0.1	0.1
	42	0.3	0.3	0.3	0.2	0.2	0.2
NO2 – N *	0	0.01	0.01	0.01	0.01	0.01	0.01
	3	0.01	0.01	0.01	0.01	0.01	0.01
	7	0.02	0.01	0.01	0.01	0.01	0.01
	14	0.01	0.01	1.44	0.01	0.01	0.88
	28	0.01	0.01	0.01	< 0.01	0.01	0.01
	42	0.01	0.01	0.01	0.01	0.01	0.01
NO3 – N *	0	0.7	0.7	0.7	0.4	0.4	0.4
	3	1.4	1.4	1.4	1.2	1.2	1.1
	7	2.2	2.2	1.9	1.4	1.4	1.5
	14	8.9	9.1	6.2	8.9	8.7	8.2
	28	9.5	9.6	9.8	8.9	8.6	8.6
	42	10.0	10.2	10.4	9.1	9.2	9.5

* Expressed as ammonium- nitrite- and nitrate-nitrogen.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the test substance has no negative effect on soil respiration, ammonification and nitrification up to a concentration of 50 mg/L of soil. Therefore, the NOEC for respiration and nitrification were both determined to be 48.3 mg a.i./kg dry soil of a loamy sand and 58.6 mg a.i./kg dry soil of a sandy loam soil.

Executive summary:

The purpose of this study was to determine the effects of the test item on soil micro-organisms by measuring the soil respiration, ammonification and nitrification in a laboratory study. For this purpose, two agricultural soils, a loamy sand (‘Collombey’) and a sandy loam (‘Strassenacker’), were incubated with the test chemical under various conditions. The test did not follow any established test guideline and was not compliant to GLP. However, the principles of the test were similar to those recommended in OECD TG 216 and 217 and the study is well documented. It is considered to be valid, reliable and suitable for hazard characterisation, with the limitation that the test concentrations were selected on the basis of typical application rates used under Good Agricultural Practice and no attempt was made to select higher concentrations being potentially inhibitory to soil micro-organisms. The test substance concentration in the soils was 5 and 50 mg/L of soil, corresponding to 4.83 and 48.3 mg/kg soil dw (Collombey) and 5.86 and 58.6 mg/kg soil dw (Strassenacker). These figures corresponded to a maximum recommended field rate of 2.5 kg a.i./ha and the ten-fold rate. The respiration test was carried out for 28 days and the nitrification test was conducted for 42 days.

Basic respiration as well as microbial respiration resulting from organic matter turn-over of the lucerne meal amended soils were not found to be significantly influenced by the high test substance concentration in either soil. This conclusion is further supported by the measurement of the ammonium, nitrite and nitrate concentrations in the soil samples at the beginning and the end of the respective experiments showing no significant negative influence of the test substance on the ammonification and nitrification process and, hence on the turn-over of organic matter in either soil. The nitrification of ammonium sulphate was investigated in detail in the 42-day study, and no negative effect of the herbicide on soil nitrification was found. Based on the findings, it can be concluded that the test substance has no negative effects on the turn-over of organic matter in the two tested soils and on the soil fertility. Therefore, the NOEC for respiration and nitrification was determined to be 48.3 mg a.i./kg dry soil in the loamy sand soil and 58.6 mg a.i./kg dry soil in the sandy loam soil.

Description of key information

42-d NOEC = 48.3 mg/kg dry soil (loamy sand soil), Nitrification, No guideline followed, Ellghausen 1985

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

48.3 mg/kg soil dw

Additional information

The purpose of this study was to determine the effects of the test item on soil micro-organisms by measuring the soil respiration, ammonification and nitrification in a laboratory study. For this purpose, two agricultural soils, a loamy sand (‘Collombey’) and a sandy loam (‘Strassenacker’), were incubated with the test chemical under various conditions. The test did not follow any established test guideline and was not compliant to GLP. However, the principles of the test were similar to those recommended in OECD TG 216 and 217 and the study is well documented. It is considered to be valid, reliable and suitable for hazard characterisation, with the limitation that the test concentrations were selected on the basis of typical application rates used under Good Agricultural Practice and no attempt was made to select higher concentrations being potentially inhibitory to soil micro-organisms. The test substance concentration in the soils was 5 and 50 mg/L of soil, corresponding to 4.83 and 48.3 mg/kg soil dw (Collombey) and 5.86 and 58.6 mg/kg soil dw (Strassenacker). These figures corresponded to a maximum recommended field rate of 2.5 kg a.i./ha and the ten-fold rate. The respiration test was carried out for 28 days and the nitrification test was conducted for 42 days.

Basic respiration as well as microbial respiration resulting from organic matter turn-over of the lucerne meal amended soils were not found to be significantly influenced by the high test substance concentration in either soil. This conclusion is further supported by the measurement of the ammonium, nitrite and nitrate concentrations in the soil samples at the beginning and the end of the respective experiments showing no significant negative influence of the test substance on the ammonification and nitrification process and, hence on the turn-over of organic matter in either soil. The nitrification of ammonium sulphate was investigated in detail in the 42-day study, and no negative effect of the herbicide on soil nitrification was found. Based on the findings, it can be concluded that the test substance has no negative effects on the turn-over of organic matter in the two tested soils and on the soil fertility. Therefore, the NOEC for respiration and nitrification was determined to be 48.3 mg a.i./kg dry soil in the loamy sand soil and 58.6 mg a.i./kg dry soil in the sandy loam soil.