Chlorothalonil

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jan 2002 to 05 Jul 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

LOAMY SAND SOIL
On receipt, the soil was refrigerated at 2 to 6°C for 94 days at a moisture content of 20.57%. Samples with dry weight equivalent to 800g were weighed into plastic boxes (18 x 18 x 9 cm) for respiration and 100g into sample pots (7.5 cm diameter x 8 cm depth) for nitrogen mineralisation. The respiration sample containers were covered with plastic lids, containing eight small holes, to prevent moisture loss. The nitrogen transformation sample pots were covered with plastic lids containing six small holes to allow gas exchange but minimise moisture loss. The soils were then incubated at a temperature of 20 ± 2°C for 10 days, prior to the start of the study.

CLAYEY SAND SOIL
On receipt, the soil was refrigerated at 2 to 6°C for 69 days at a moisture content of 16.21%. Samples with dry weight equivalent to 800g were weighed into plastic boxes (18 x 18 x 9cm) for respiration and samples with dry weight equivalent to lOOg were weighed into sample pots (7.5cm diameter x 8cm depth) for nitrogen mineralisation. The respiration sample containers were covered with plastic lids, containing eight small holes, to allow gas exchange but minimise moisture loss. The nitrogen transformation sample pots were covered with plastic lids containing six small holes to allow gas exchange but minimise moisture loss. The soil was then incubated at a temperature of 20 ± 2°C for 14 days prior to the start of the study.

APPLICATION OF THE TEST SUBSTANCE
Mode of addition of test substance to soil was done by preparing stock solutions by diluting the following amounts of the test substance to 500 mL with distilled water:
- 8 mg a.i./kg: 0.7533g
- 12 mg a.i./kg: 1.1300g
10 mL of each of these dilutions was distributed per kg soil (dry weight). This was equivalent to nominal application rates of 8mg a.i./kg and 12 mg a.i./kg (assuming 100% distribution in soil to a depth of 5 cm, with a soil bulk density of 1.5 g/cm3).

Test organisms (inoculum):

soil

Total exposure duration:

98 d

Remarks:

98 days for the nitrification test and 28 days for the respiration test

Test temperature:

20 ± 2°C

Moisture:

45 ± 5% WHC

Organic carbon content (% dry weight):

3.3

Nitrogen content (% dry weight):

0.225

Details on test conditions:

TEST SYSTEM SPECIFICATIONS
- Treatment replication: 6 x 1000g or 66 x 100g dry weight equivalent soil replicates per treatment for respiration determination and nitrogen transformations, respectively.
- Glucose amendment concentrations employed in respiration determinations:
- Loamy sand soil: 0.6g/100g soil dw
- Clayey sand soil: 0.4g/100g soil dw
- Lucerne meal: Dried lucerne, ground to pass through a 0.5mm sieve, containing organic carbon (34.4%) and total nitrogen (2.5%) with a C:N ratio of 14:1
- pH analysis: Volumes (10ml) of sieved, air dried soil were added to 50ml of water, left to shake for 5 minutes and then left to stand for between 2 and 24 hours. The pH of the soil was then determined in the supernatant.

SOIL CHARACTERISTICS - LOAMY SAND SOIL
The loamy sand soil was obtained from a supplier on 19th October 2001. The soil was collected from a grassland site that had not received pesticide or fertiliser treatments within the previous 12 months. The soil was received, stored prior and during analysis in a manner that best suited the analytical requirements. The soil was collected fresh from the field on 17th October 2001 and air-dried sufficiently to allow sieving through a 2mm sieve. Each soil was then thoroughly mixed, subsampled and delivered to the testing facility. The sub-sample was fully characterised (see Table 1 in any other information on materials and methods incl. tables).

SOIL CHARACTERISTICS - CLAYEY SAND SOIL
The clayey sand soil was obtained from Agronomy Enterprise on 13th November 2001. The soil was collected from a grassland site that had not rece ived pesticide or fertiliser treatment within the previous 12 months. The soil collection was carried out to the principles of ISO/DIS 10381-6, Soil Quality - Sampling Part 6. The soil was collected fresh from the field on 3rd November 2001 and air dried sufficiently to allow sieving through a 2mm sieve. The soil was then thoroughly mixed and delivered to the testing facility. A sub-sample was fully characterised (see Table 1 in any other information on materials and methods incl. tables).

Replicate batches of conditioned soil were treated with the test formulation at the treatment rates of 8mg a.i./kg and 12mg a.i./ by even distribution of distilled water (containing the correct amount of Bravo 720) over the soil surface of each replicate, followed by thorough mixing. The control replicates were treated in a similar manner with distilled water alone. All replicates were adjusted to the moisture content as shown above. All treatments had lids replaced and were then incubated under aerobic conditions at 20 ± 2°C.

SOIL MICROFLORA RESPIRATION SYSTEM
At day zero (within 6 hours of addition of the test substance) and after 7, 14 and 28 days, 100g samples (dry weight equivalent) were removed for determination of microbial respiration using the glucose amendment method of Anderson and Domsch (1978). Prior to removal of samples, the weight of each soil replicate was recorded and used to determine moisture loss during the study. This information was then used to remoisturise the soil replicates to 45 ± 5% WHC as required during the course of the study. The optimum glucose amendment level was determined by amending replicate soil samples with differing levels of glucose e.g. 0.0, 0.1, 0.2, 0.4 and 0.6g and by measuring CO2 evolution overnight. The glucose amendment level giving the maximum initial respiration rate (the lowest point or plateau prior to increased rate of CO2 production) was then chosen for subsequent respiration studies on the soils tested. Carbon dioxide evolution was measured using an infra-red gas analyser at a flow rate through the detection cells of 2 litres/h. The raw data was captured using a data acquisition board and ADC gas logging software. The total CO2 evolved over a 12-hour period was calculated for each replicate. This data was then used to obtain the respiration rate by converting ppm CO, by volume for each replicate to ml CO2/h/100g soil. Soil microbial biomass was determined at time zero for each soil by the Anderson and Domsch substrate induced respiration method. The biomass value was then compared to that of the soil organic carbon value.

SOIL NITROGEN TRANSFORMATION SYSTEM
Replicate batches of conditioned soil were amended with ground lucerne (0.5% w/w) and were treated with test formulation at the treatment rates of 8 mg a.i./kgand 12 mg a.i./kg by even distribution of distilled water (containing the correct amount of test formulation) over the soil surface of each replicate, followed by thorough mixing. The control replicates were treated in a similar manner with deionised water alone. All replicates were adjusted to the moisture content as shown on previous page. All treatments were then covered with plastic lids containing six small holes to allow gas exchange and were then incubated at 20 ± 2°C.

At day 0 (within 3 hours of addition of the test substance) and after 7, 14 and 28 days, samples were removed for determination of ammonium, nitrate and nitrite-nitrogen. The clayey sand soil was also sampled in the same way at days 42, 56, 70, 84 and 98. Prior to removal of samples, the weight of each soil replicate was recorded and used to determine moisture loss during the study. This information was then used to remoisturise the soil replicates to 45 ± 5%WHC as required during the course of the study.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 8 and 12 mg a.i./kg soil dw.

Reference substance (positive control):

yes

Remarks:

Dinoseb (98%)

Key result

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

12 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

12 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Details on results:

SOIL MICROBIAL BIOMASS
The soil microbial biomass at the start of the study, as determined by the Anderson and Domsch glucose amendment method is given in 'Any other information on results incl. tables'. The results for the loamy sand and clayey sand soils were 0.049 and 0.035g C/100g soil respectively. These results should be not less than 1% of the total soil organic carbon content. The soils used in this study had total soil organic carbon contents of 2.1% w/w (loamy sand soil) and 3.3% w/w (clayey sand soil). Thus, 1% of each total gives values of 0.021 and 0.033g carbon/100 g soil dry weight with the microbial biomasses being determined being greater than this value (2.25% and 1.07% of organic carbon values, loamy sand soil and clayey sand soil, respectively). Therefore, the two soils were considered viable and acceptable for use in this study.

SOIL MICORFLORA RESPIRATION
The results on the respiration are presented in 'An other information on results incl. tables'.

The test formulation, when applied to a high organic matter loamy sand soil, at time zero, did not give rise to any statistically significant effects greater than ±17% when compared to control values. Percentage deviations of -9.35% and -16.24% were observed at the 8mg a.i./kg and 12mg a.i./kg treatment rates respectively. After 7 days incubation, no statistically significant percentage deviations in excess of ±21% were observed with respect to control values. Percentage deviations of -12.96% (8 mg a.i./kg treatment) and +20.50% (12 mg a.i./kg treatment) were observed. At day 14, no statistically significant effects in excess of ±14% were observed. Percentage deviations were found to be -6.38% (8mg a.i./kg treatment) and -13.98% (12mg a.i./kg treatment). At day 28, a statistically significant deviation not in excess of ±25% was observed at the 8mg a.i./kg treatment rate. Percentage deviations of +5.57% and -0.52% were observed at the 8mg a.i./kg and 12mg a.i./kg treatment rates respectively. There was no observed effect on soil pH at the end of the study. The control replicates meet the ±15% variation criteria.

NITRATE FORMATION RATES
The results on the respiration are presented in 'An other information on results incl. tables'.

Nitrate formation rates were calculated between each time period (i.e. 0-7, 7-14 days etc.) and the overall rate using the linear part of the figure. Percentage deviations from control rates were compared. The overall rate of nitrate formation was calculated for each treatment, using the linear part of the graph. The overall rate for the formation of nitrate between 7 and 28 days is 1.93 mgN/kg soil/day for the control treatment whilst for the 8 mg a.i./kg treatment rate it is 1.92 mg N/kg soil/day representing a non-significant percentage deviation of -0.44% from the control rate. The rate of nitrate formation at the higher rate is 2.22 mg N/kg soil/day, representing a non-significant percentage deviation of +15.38% from the control values. There was no observed effect on soil pH at the end of the study. The reference substance (Dinoseb acetate) produced significant effect on soil nitrogen transformation processes in soils of the type commonly used in these studies.

The test formulation, when applied to a low organic matter clayey sand soil at 8 mg a.i/kg and 12 mg a.i./kg, did not give rise to any statistically significant deviations on nitrate concentrations, at time zero. Percentage deviations of +3.87% and +7.33% were observed at the 8 mg a.i/kg and 12 mg a.i./kg treatments respectively. Low levels of ammonium were observed in all treatments. After 7 days incubation, statistically significant percentage deviations greater than ±25% were observed for nitrate concentrations at both treatment rates. Percentage deviations of +45.95% and +44.90% were observed at the 8 mg a.i/kg and 12 mg a.i./kg treatment rates respectively. Ammonium levels were found to be below the limit of quantitation and remained so for the rest of the study. At day 14, statistically significant percentage deviations greater than ±25% were observed at both treatment rates for nitrate concentrations. A percentage deviation of +38.15% was observed at the 8 mg a.i/kg treatment rate. A percentage deviation of +42.70% was observed at the 12 mg a.i/kg treatment rate. After 28 days incubation, a statistically significant deviation in excess of ±25% was observed at the higher treatment rate only. A percentage deviation of +5.25% was determined for the 8 mg a.i/kg treatment rate. The effect determined on nitrate concentrations up until this timepoint was therefore determined to be transient. A percentage deviation of +35.94% was observed for nitrate concentrations at the 12 mg a.i./kg treatment rate.

After 42 days incubation, a statistically significant deviation in excess of ±25% was observed at the 12 mg a.i./kg treatment rate. Percentage deviations of +14.97% and +31.07% were observed at the 8 mg a.i/kg and 12 mg a.i./kg treatment rates respectively, with regard to nitrate concentrations. At day 56, a statistically significant deviation was observed at both treatment rates, with regard to nitrate concentrations. At the 8 mg a.i/kg treatment rate a deviation of +24.22% was observed. At the 12 mg a.i/kg treatment rate a percentage deviation of +43.03% was observed. At day 70, a statistically significant deviation not in excess of ±25% was observed for nitrate concentrations at the 8 mg a.i/kg treatment rate. A statistically significant deviation in excess of ±25% was observed at the higher treatment rate. Percentage deviations at the 8 mg a.i/kg and 12 mg a.i/kg treatment rates were found to be +22.13% and +44.33% respectively. After 84 days incubation, a statistically significant deviation was observed for nitrate concentrations at the higher treatment rate only. Percentage deviations at the 8 mg a.i/kg and I 2 mg a.i/kg treatment rates were found to be +9.47% and +43.72% respectively. At day 98, no percentage deviations in excess of ±25% was observed for nitrate concentrations. At the 8 mg a.i/kg treatment rate a percentage deviation of +0.11% was observed. At the 12 mg a.i/kg treatment rate a statistically significant percentage deviation of +22.97% was observed. The effect observed at the higher treatment rate was therefore determined to be transitory. The control nitrate replicates met the ±15% variation criteria at all time points with the exception of days 56 and 84. At these two time points the RSD for the control inter-replicate variation was in excess of ±15%.

Nitrate formation rates were calculated between each time period (i.e. 0-7, 7-14 days etc.) and the overall rate using the linear part of the figure. Percentage deviations from control rates were compared. The overall rate of nitrate formation was calculated using the linear part of the graph. The overall rate for the formation of nitrate between 7 and 98 days is 0.45 mgN/kg soil/day for the control treatment whilst for the 8 mg a.i./kg treatment it is 0.45 mgN/kg soil/day representing a deviation of 0.00% from the control rate. The rate of nitrate formation in the 12 mg a.i./kg treatment was higher than in the control and 8 mg a.i./kg soils, with a value of 0.59 mgN/kg soil/day. This represents a non-significant percentage deviation from the control of +29.72%. There was no observed effect on soil pH at the end of the study. The reference substance (Dinoseb acetate) produced significant effect on soil nitrogen transformation processes in soils of the type commonly used in these studies.

Results with reference substance (positive control):

The reference substance (Dinoseb acetate) produced significant effect on soil nitrogen transformation processes in soils of the type commonly used in these studies.

Reported statistics and error estimates:

RESPIRATION
The data were statistically analysed by analysis of variance. Where a statistically significant difference between treatments was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment at the 5% level. Percentage deviation with respect to control values were also calculated and compared against ±25% effect criteria.

NITROGEN TRANSFORMATION
The concentration data was statistically analysed by analysis of variance. Where a statistically significant difference between treatments was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment at the 5% level. Nitrate formation rates were calculated for each treatment, between each time point and an overall rate calculated. Percentage deviations with respect to control values were calculated for both concentration and rate data and compared with ±25% effect criteria given in the guideline.

 Table 2: Effect of the test formulation on mean microbial respiration rate (mg CO2/h/kg soil) in a loamy sand soil

Treatment	mg CO2/h/kg soil
	Day 0	Day 7	Day 15	Day 28
Control	33.06	30.39	25.56	24.36
8 mg a.i./kg	29.97 (-9.35)	26.45 (-12.96)	23.93 (-6.38)	25.71 (+5.57)*
12 mg a.i./kg	27.69 (-16.24)	24.16 (-20.50)	21.99 (-13.98)	24.23 (-0.52)
F-ratio	25.310	11.715	6.708	4.779

Figures are means of three replicates

( ) = % Variation from control treatment

F Ratio = Ratio of (SST x k(n-1))/(SSE x (k-1))

 

 

Table 3: Effect of the test formulation on mean microbial respiration rate (mg CO2/h/kg soil) in a clayey sand soil

Treatment	mg CO2/h/kg soil
	Day 0	Day 7	Day 15	Day 28
Control	42.23	71.76	41.76	37.22
8 mg a.i./kg	39.07(-7.47)	40.82(-2.16)	40.35(-3.40)	35.56 (-4.46)
12 mg a.i./kg	36.09(-14.52)	35.89(-14.06)	35.89(-14.06)	30.99 (-16.74)
F-ratio	16.236	11.179	8.226	6.693

Figures are means of three replicates

( ) = % Variation from control treatment

F Ratio = Ratio of (SST x k(n-1))/(SSE x (k-1))

 

Table 4: Effect of the test formulation on mean NH/-N, NO3-N and total mineral nitrogen

(N-min ) concentrations (mg N/kg soil) in a loamy sand soil

Treatment		mg N/kg soil (and % variance from control groups)
		Day 0		Day 7		Day 14		Day 28
Control	NH4+	3.90		<0.53		<0.53		<0.53
	NO3-	102.23		103.89		128.20		144.35
	NO2	<0.11		<0.11		<0.11		<0.11
	N-min	106.13		103.89		128.20		144.35
Low rate	NH4+	3.02	-22.56*	4.32	ND	<0.53	ND	<0.53	ND
	NO3-	95.70	-6.39	108.86	+78	120.96	-5.65	149.14	+3.32
	NO2	<0.11	ND	<0.11	ND	<0.11	ND	<0.11	ND
	N-min	98.72	-6.98	113.23	+8.99	120.94	-5.65	149.14	+3.32
High rate	NH4+	1.77	-54.62*	3.92	ND	<0.53	ND	<0.53	ND
	NO3-	95.57	-6.51	105.41	+1.46	123.94	-3.32	152.08	+5.36*
	NO2	<0.11	ND	<0.11	ND	<0.11	ND	<0.11	ND
	N-min	97.34	-8.28	109.38	+5.28	123.94	-3.32	152.08	+5.36*
F-ratio	NH4+	59.591		ND		ND		ND
	NO3-	2.794		0.580		3.238		2.579
	NO2	ND		ND		ND		ND
	N-min	0.288		1.584		3.238		2.579

Figures are means of four replicates

F Ratio = Ratio of sum of squares ( treatment ) / sum of squares (error)

- LOQ NH4+: 0.53 mg/kg

- LOQ NO3-: 1.06 mg/kg

- LOQ NO2: 0.11 mg/kg

ND = not determined

*) significant (at p = 0.05)

N-min = Total mineralised nitrogen (i.e. total of ammonium + nitrate + nitrite (if present)

 

 

Table 5: Mean rate of Nitrate formation (mg NO3-/kg soil/dav) in a loamy sand soil treated with the test formulation

Treatment	mg N/kg soil/day
	0-7 Days	7-14 Days	14-28 Days	Overall rate (7-28 Days)
Control	0.24	3.47	1.15	1.93
Low rate	1.88 (+691.01)	1.73 (-50.22)	2.01 (+74.50)	1.92 (-0.44)
High rate	1.41 (+491.53)	2.65 (-23.75)	2.01 (+74.28)	2.22 (+15.38)

( ) = % Variation from control treatment

 

Table 6: Effect of the test formulation on mean NH4+ -N, NO3- -N, NO2 -N and total minral nitrogen (N-min) concentrations (mg N/kg soil) in a clayey sand soil

Treatment		mg N/kg soil (and % variance from control groups)
		Day 0		Day 7		Day 14		Day 28
Control	NH4+	3.90		<0.53		<0.53		<0.53
	NO3-	102.23		103.89		128.20		144.35
	NO2	<0.11		<0.11		<0.11		<0.11
	N-min	106.13		103.89		128.20		144.35
Low rate	NH4+	3.02	-22.56*	4.32	ND	<0.53	ND	<0.53	ND
	NO3-	95.70	-6.39	108.86	+78	120.96	-5.65	149.14	+3.32
	NO2	<0.11	ND	<0.11	ND	<0.11	ND	<0.11	ND
	N-min	98.72	-6.98	113.23	+8.99	120.94	-5.65	149.14	+3.32
High rate	NH4+	1.77	-54.62*	3.92	ND	<0.53	ND	<0.53	ND
	NO3-	95.57	-6.51	105.41	+1.46	123.94	-3.32	152.08	+5.36*
	NO2	<0.11	ND	<0.11	ND	<0.11	ND	<0.11	ND
	N-min	97.34	-8.28	109.38	+5.28	123.94	-3.32	152.08	+5.36*
F-ratio	NH4+	59.591		ND		ND		ND
	NO3-	2.794		0.580		3.238		2.579
	NO2	ND		ND		ND		ND
	N-min	0.288		1.584		3.238		2.579

 

Treatment		mg N/kg soil
		Day 0	Day 7	Day 14	Day 28	Day 42	Day 56	Day 70	Day 84	Day 98
Control	NH4+	6.38	<0.53	<0.53	<0.53	<0.53	<0.53	<0.53	<0.53	<0.53
	NO3-	19.65	22.94	27.94	34.28	37.82	50.03	55.77	45.50	64.04
	NO2	0.29	<0.11	<0.11	<0.11	<0.11	<0.11	<0.11	<0.11	<0.11
	N-min	26.32	22.94	27.94	34.28	37.82	50.03	55.77	45.50	64.04
Low rate	NH4+	5.78 (-9.40)	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND
	NO3-	20.41 (+3.87)	33.48 (+45.95)*	38.60 (+38.15)*	36.08 (+5.25)	43.48 (+14.97)	56.72 (+24.22)*	61.10 (+22.13)*	61.05 (+9.47)	64.11 (+0.11)
	NO2	0.35 (+20.69)*	<0.11 ND	<0.11 ND	<0.53 ND	<0.11 ND	0.20 ND	<0.11 ND	<0.11 ND	<0.11 ND
	N-min	26.54 (+0.84)	33.48 (+45.95)*	38.60 (+38.15)*	36.08 (+5.25)	43.48 (+14.97)	56.72 (+24.66)*	61.10 (+22.13)*	61.05 (+9.47)	64.11 (+0.11)
High rate	NH4+	6.01 (-5.80)	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND	<0.53 ND
	NO3-	21.09 (+7.33)	33.24 (+44.90)*	39.87 (+42.70)*	46.60 (+35.94)*	49.57 (+31.07)*	65.08 (+43.03)*	72.21 (+44.33)*	80.15 (+43.72)*	78.75 (+22.97)*
	NO2	0.35 (+20.69)*	<0.11 ND	<0.11 ND	<0.11 ND	<0.11 ND	<0.11 ND	<0.11 ND	<0.11 ND	<0.11 ND
	N-min	27.45 (+4.29)	33.24 (+44.90)*	39.87 (+42.70)*	46.60 (+35.94)*	49.57 (+31.07)*	65.20 (+43.30)*	72.21 (+44.33)*	80.15 (+43.72)*	78.75 (+22.97)*
F-ratio	NH4+	6.962	ND	ND	ND	ND	ND	ND	ND	ND
	NO3-	0.615	21.418	53.774	4.812	8.277	11.336	10.680	18.004	4.036
	NO2	3.500	ND	ND	ND	ND	ND	ND	ND	ND
	N-min	0.355	21.418	53.774	4.812	8.277	11.549	10.68	18.004	4.036

 

 

Table 7: Mean rate of Nitrate formation (mg NO3/kg soil/day) in a test formulation treated clayey sand soil

Treatment	mg N/kg soil/day
	0-7 Days	7-14 Days	14-28 Days	28-42 Days	42-56 Days	56-70 Days	70-84 Days	84-98 Days	Overall rate 0-98 Days
Control	0.47	0.71	0.45	0.25	0.55	0.32	0.41	0.59	0.45
8 mg a.i./kg	1.87 (+297.07)	0.73 (+2.35)	-0.18 (-139.82)	0.53 (+108.20)	0.93 (+70.04)	0.33 (+1.24)	0.00 ND	0.22 (-63.12)	0.45 (0.00)
12 mg a.i./kg	1.72 (+266.49)	0.95 (+32.43)	0.48 (+6.50)	0.21 (-16.77)	1.11 (+102.19)	0.51 (+57,64)	0.57 (+38.63)	-0.10 (-116.97)	0.59 (+29.72)

( ) = % Variation from control treatment

 

Validity criteria fulfilled:

yes

Conclusions:

This study is designed to detect long-term potential effects of plant protection products on soil respiration and nitrogen transformation processes. The data presented in this report concludes that the test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and low organic matter clayey sand soil would not adversely effect soil microbial respiration. The test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and at 8mg/kg to a low organic matter clayey sand soil would not adversely effect soil microbial nitrogen transformation processes. Therefore, the 28-d NOEC was determined to be ≥ 12 mg/kg.

Executive summary:

The influence of the test formulation, a SC formulation of the test substance, on soil microflora was determined using short term respiration experiments and by monitoring nitrogen transformation in accordance with OECD TG 216 and 217. In addition, the study was performed using two soils, as described in the Dutch Standard NEN 5795, in order to provide for a wide applicability of the results obtained. A dispersion of The test formulation in distilled water was applied to two soils, one a high organic matter loamy sand soil and the other a low organic matter clayey sand soil at a nominal application rate of 8 mg/kg The test formulation. The test was also conducted at 12 mg/kg. The treatment rates were based on predicted environmental concentration calculations, taking into account the short half-life of the test substance. The effects of The test formulation on microbial respiration were investigated using short-term respiration experiments conducted after 0, 7, 14 and 28 days. On each occasion, aliquots of soil were amended with a non-limiting quantity of glucose and carbon dioxide evolution measured over the subsequent 24 hour period using an infra-red gas analyser. Values for substrate induced respiration rate were converted to soil microbial respiration (mg CO2/h/kg soil) for data presentation. The effects on nitrogen transformation, ammonification and nitrification were investigated by determining ammonium-N, nitrate-N and nitrite-N concentrations in soil amended with ground lucerne grass. Aliquots of soil were extracted with 2M KCl after 0, 7, 14, and 28 days and after 0, 7, 14, 28, 42, 56, 70, 84 and 98 days. The concentrations of inorganic nitrogen species in the extracts were determined colour metrically

When applied to a high organic matter loamy sand soil at 8 mg/kg and at 12 mg/kg, the test formulation did not give rise to any statistically significant deviations, in excess of ±25% during the course of the study. After 28 days incubation all deviations from control values were within ±6%. The test formulation, when applied to a low organic matter clayey sand soil at 8 mg/kg and at 12 mg/kg, did not give rise to any statistically significant deviations, in excess of ±25% during the course of the study. After 28 days incubation all deviations from control values were within ±17%.When applied to a high organic matter loamy sand soil at 8 mg/kg and at 12 mg/kg, The test formulation did not cause any statistically significant long term effects on nitrogen transformation processes based on assessment of nitrate concentrations. After 28 days incubation the concentrations of nitrate measured in each treatment rate were found to be within ± 6% of the control values. The overall rate of formation of nitrate did not result in a statistically significant difference exceeding ± 25% variation from control values. 

The test formulation, when applied to a low organic matter clayey sand soil at 8 mg/kg and at 12 mg/kg had a transitory effect on nitrate concentrations. At the 8 mg/kg treatment rate this effect could be observed up to and including day 14. At day 28 and for the remainder of the study no statistically significant deviations in excess of ± 25% were observed at this treatment rate. This stimulatory effect was observed up until and including day 84 in the 12 mg/kg treatment rate. After 98 days incubation, no statistically significant deviations in excess of ± 25% were observed for nitrate concentrations. The overall rate of nitrate formation did not result in any statistically significant deviations. At the 8 mg/kg treatment rate a percentage deviation of 0.00% was observed. At the 12 mg/kg treatment rate a percentage deviation of 29.72% was observed.

The data presented in this report concludes that the test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and low organic matter clayey sand soil would not adversely affect soil microbial respiration. The test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and at 8 mg/kg to a low organic matter clayey sand soil would not adversely affect soil microbial nitrogen transformation processes. Therefore, the 28-d NOEC was determined to be ≥ 12 mg/kg.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sep 2000 to 01 Nov 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

A low rate working stock solution was prepared by diluting the following amounts of each metabolite to 1 litre with distilled water:
- Low rate additions: 0.06 g M4, 0.03 g M5 and 0.04 g M9.

A high rate working stock solution was prepared by diluting the following amounts of each metabolite to 50mL with distilled water:
- High rate additions: 0.024 g M4, 0.0142 g M5 and 0.0167 g M9

10mL of the working stock solutions were distributed per kg soil ( dry weight ). This was equivalent to nominal application rates of (assuming 100% distribution in soil to a depth of 5 cm, with a soil bulk density of 1.5g/cM5):
- Low rate: 0.6 mg M4, 0.3 mg M5 and 0.4 mg M9.
- High rate: 4.8 mg M4, 2.8 mg M5 and 3.3 mg M9.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

45 ± 5% WHC

Organic carbon content (% dry weight):

1.2

Nitrogen content (% dry weight):

0.19

Details on test conditions:

TEST SYSTEM SPECIFICATIONS
- Treatment replication: 3 x 1000g or 40 x 50g dry weight equivalent soil replicates per treatment for respiration determination and nitrogen transformations, respectively.
- Glucose amendment concentrations employed in respiration determinations: For silty sand soil , 0.6g/100g soil (dry weight).
- Lucerne meal: Dried lucerne, ground to pass through a 0.5mm sieve, containing organic carbon (34.4%) and total nitrogen (2.5%) with a C:N ratio of 14:1
- pH analysis: Volumes (10mL) of sieved, air dried soil were added to 50ml of water, left to shake for 5 minutes and then left to stand for between 2 and 24 hours. The pH of the soil was then determined in the supernatant.

SOIL CHARACTERISTICS
The soil used in this study was obtained from Levington Agriculture Limited . The soil collected from a grassland site that had not received pesticide or fertiliser treatments within the previous 12 months. The soil was collected and prepared according to the international standards ISO/DIS 10381-6, Soil Quality - Sampling Part 6. The soil was collected fresh from the field on 31 July 2000 and air dried sufficiently to allow sieving through a 2mm sieve. The soil was then thoroughly mixed, sub-sampled and delivered to Chemex International plc on 2 August 2000 . The sub-sample was fully characterised. See ‘Any other information on materials and methods incl. tables’. On receipt, the soil was refrigerated at 2 to 6 °C for 14 days at a moisture content of 15.72%. Samples with dry weight equivalent to 1000 g and 50g were weighed into plastic boxes ( IS x 18 9cm) for respiration and into sample pots (6 cm diameter x 7 cm depth ) for nitrogen mineralisation . The respiration sample containers were covered with plastic lids to prevent moisture loss and the nitrogen transformation sample pots were covered with plastic lids containing four small holes to allow gas exchange but minimise moisture loss. The soil was then incubated at a temperature of 20 ± 2°C for 42 days prior to the start of the study.

SOIL MICROFLORA RESPIRATION SYSTEM
Replicate batches of conditioned soil were treated with a low rate (0.6 mg/kg M4, 0.3 mg/kg M5, 0.4 mg/kg M9) and a higher rate (4.8 mg/kg M4, 2.8 mg/kg M5, 3.3 mg/kg M9) of the test substance by even distribution of distilled water (containing the correct application rate of the parent substance) over the soil surface of each replicate, followed by thorough mixing. The control replicates were treated in a similar manner with distilled water alone. All replicates were adjusted to the moisture content. All treatments had lids replaced and were then incubated under aerobic conditions at 20 ± 2°C . At day zero (within 3 hours of addition of the test substance) and after 7, 15 and 28 days, 100g samples (dry weight equivalent) were removed for determination of microbial respiration using the glucose amendment method of Anderson and Domsch (1978). Prior to removal of samples, the weight of each soil replicate was recorded and used to determine moisture loss during the study. This information was then used to remoistunse the soil replicates to 45 ± 5%WHC as required during the course of the study. The optimum glucose amendment level was determined by amending replicate soil samples with differing levels of glucose e.g. 0.0, 0.1, 0.2, 0.4 and 0.6g and by measuring CO2 evolution overnight. The glucose amendment level giving the maximum initial respiration rate (the lowest point or plateau prior to increased rate of CO2 production) was then chosen for subsequent respiration studies on the soils tested. Carbon dioxide evolution was measured using an infrared gas analyser at a flow rate through the detection cells of 2 litres/h. The raw data was captured using a data acquisition board and ADC gas logging software. The total CO2 evolved over a 12-hour period was calculated for each replicate. This data was then used to obtain the respiration rate by converting ppm CO2 by volume for each replicate to mg CO2/h/kg soil. Soil microbial biomass was determined at time zero for each soil by the Anderson and Domsch substrate induced respiration method. The soil biomass carbon was then compared to that of the soil organic carbon value.

SOIL NITROGEN TRANSFORMATION SYSTEM
To initiate the test, replicate batches of conditioned soil were amended with ground lucerne (0.5% w/w) and treated with the low rate and the higher rate of the test substance by even distribution of distilled water (containing the correct application rate of the metabolites) over the soil surface of each replicate, follow ed by thorough mixing. The control replicates were treated in a similar manner with distilled water alone. All treatments were then covered with plastic lids containing four small holes to allow gas exchange. At day 0 (within 3 hours of addition of the test substance) and after 7, 14 and 28 days, samples were removed for determination of ammonium, nitrate and nitrite-nitrogen. Prior to removal of samples, the weight of each soil replicate was recorded and used to determine moisture loss during the study. This information was then used to remoisturise the soil replicates to 45 ± 5 % WHC as required during the course of the study.

Nominal and measured concentrations:

The test substance was applied to soil at two rates, one a low rate and the other a high rate. The low rate was equivalent to a nominal application rate of 0.6 mg/kg M4, 0.3 mg/kg M5 and 0.4 mg/kg M9. The higher application rate was equivalent to 4.8 mg/kg M4, 2.8 mg/kg M5 and 3.3 mg/kg M9.

Reference substance (positive control):

yes

Remarks:

Dinoseb acetate

Key result

Duration:

28 d

Dose descriptor:

ER25

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Remarks on result:

other: high application rate of multiple metabolites

Key result

Duration:

28 d

Dose descriptor:

ER25

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: high application rate of multiple metabolites

Details on results:

All results are presented in ‘Any other information on results incl. tables’.

SOIL MICROBIAL BIOMASS
The result for the soil was 0.043g C/100g soil. This result should be not less than 1% of the total soil organic carbon content. The soil used in this study had a total soil organic carbon content of 1.2% w/w. Thus, 1% of the total gives a value of 0.012g carbon/100g soil dry weight with the microbial biomass determined being greater than this value (3.58% of organic carbon values). Therefore, the soil was acceptable for use in this study.

SOIL MICROFLORA RESPIRATION
The metabolites, when applied to a low organic matter silty sand soil, at zero time, did not give rise to any statistically significant effects. A percentage deviation of -6.33% was observed for the lower rate whilst a deviation of -1.76% was observed at the higher rate. After 7 days incubation, no statistically significant effects were observed . Percentage deviations of -0.52% and -1.31% were observed at the lower rate and higher rate respectively. At day 15, percentage deviations of -2.45% and -3.53% were observed at the lower and higher rates respectively, with no statistical significance observed at either application rate. After 28 days incubation, no statistically significant effects were observed at either application rate. Percentage deviations were -1.26% and +2.68% for the lower and higher rates respectively. There was no observed effect on soil pH at the end of the study.

SOIL NITROGEN TRANSFORMATION
- Mineral Nitrogen Concentrations: The metabolites, when applied to a low organic matter silty sand soil at time zero , did not give rise to any statistically significant effects on nitrate concentrations. At the lower rate a percentage deviation of -3.45% (nitrate) was found, whilst at the higher rate, a percentage deviation of -3.94% (nitrate), was observed. After 7 days incubation, no statistically significant effects were observed at the lower rate, with a percentage deviation of -7.27% observed for nitrate concentrations. At the higher rate application, no statistically significant effect was observed on nitrate concentrations. A percentage deviation of -0.72% was observed. At day 14 no statistically significant effects were observed on nitrate concentrations at either the higher or lower application rates. A percentage deviation of -5.30% was observed for nitrate concentrations at the lower application rate, whilst at the higher application rate a deviation of -1.36% was observed. After 28 days incubation no statistically significant effects were observed at either application rate. A percentage deviation of -4.80% was observed for nitrate concentrations at the Lower application rate, whilst at the higher application rate a percentage deviation of +4.18% was observed for nitrate concentrations.

- Nitrogen formation rates: Nitrate formation rates were calculated between each time period ( i.e. 0-7, 7-14 days etc. ) and the overall rate using the linear part of the figure. Percentage deviations from control rates were compared. Between 0 and 7 days a percentage deviation from control values of -46.67% was observed at the low rate, whilst at the high rate a percentage deviation of-33.33% was calculated . Between days 7 and 14, percentage deviations from control rates were found to be -0.61 % for the low rate and -8.48% for the high rate applications. Between 14 and 28 days percentage deviations between treatment and control rates were found to be -3.62% at the low rate and +10.87% at the high application rate. The overall rate of nitrate formation was calculated for each treatment, using the linear part of the graph. The overall rate for the formation of nitrate between 7 and 28 days is 1.47mgN/kg soil/day for the control treatment whilst for the low' rate it is 1.44mgN/kg soil, day representing a deviation of -2.04% from the control rate. The rate of nitrate formation at the high rate is 1.62mgN7kg soil/day, representing a deviation of -10.20% from the control values. Neither of the deviations was found to be statistically significant. There was no observed effect on soil pH at the end of the study.

Results with reference substance (positive control):

The reference substance (Dinoseb acetate) produced a significant effect on soil microflora respiration in soils of the type commonly used in these studies.Furthermore, the reference substance (Dinoseb acetate) produced significant effect on soil nitrogen transformation processes in soils of the type commonly used in these studies.

Reported statistics and error estimates:

- Respiration: The data were statistically analysed by analysis of variance. Where a statistically significant difference between treatments was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment at the 5% level. Percentage deviation with respect to control values were also calculated and compared against =25% effect criteria.
- Nitrogen Transformation: The concentration data was statistically analysed by analysis of variance. Where a statistically significant difference between treatments was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment at the 5% level. Nitrate formation rates were calculated for each treatment, between each time point and an overall rate calculated . Statistical analysis of the overall rate data (7-28 days ) was undertaken by subtracting the mean nitrate concentration for each treatment at day 7, from the values after 28 days. The resulting concentrations were converted into rates and the data analysed as described as above. Percentage deviations with respect to control values were calculated for both concentration and rate data and compared with = 25% effect criteria given in the guideline. Statistical analyses were conducted using Toxstat Release 3.0 (1989).

Table 2: Effect of the test substance’ metabolites on mean microbial respiration rate (mg CO2/h/kg soil) in a silty sand soil

Treatment	mg CO2/h/kg soil
	Day 0	Day 7	Day 15	Day 28
Control	48.18	45.72	44.53	40.33
Low rate	45.13 (-6.33)	45.96 (+0.52)	43.44 (-2.45)	40.84 (-1.26)
High rate	47.33 (-1.76)	45.12 (-1.31)	42.96 (-3.53)	41.41 (-2.68)
F-ratio	2.79	0.264	0.388	1.043

Figures are means of three replicates

( ) = % Variation from control treatment

F Ratio = Ratio of (SST x k(n-1))/(SSE x (k-1))

 

Table 3: Effect of the test substance’ metabolites on mean NH/-N, NO3-N and total mineral nitrogen

(N-min ) concentrations (mg N/kg soil) in a silty sand soil

Treatment		mg N/kg soil (and % variance from control groups)
		Day 0		Day 7		Day 14		Day 28
Control	NO2	0.12		<0.10		<0.10		<0.10
	NO3-	33.00		36.18		47.75		67.03
	NH4+	10.25		<0.26		<0.26		<0.26
	N-min	43.37		36.18		47.75		67.03
Low rate	NO2	0.15	+25.00	<0.10	ND	<0.10	ND	<0.10	ND
	NO3-	31.86	-3.45	33.55	-7.27	45.22	-5.30	63.81	-4.80
	NH4+	9.95	-2.93	<0.26	ND	<0.26	ND	<0.26	ND
	N-min	41.96	-3.25	33.55	-7.27	45.22	-5.30	63.81	-4.80
High rate	NO2	0.13	+8.33	<0.10	ND	<0.10	ND	<0.10	ND
	NO3-	31.70	-3.94	35.92	-0.72	48.4	-1.36	69.83	-4.18
	NH4+	8.30	-19.02	<0.26	ND	<0.26	ND	<0.26	ND
	N-min	40.13*	-7.47	35.92	-0.72	48.4	-1.36	69.83	-4.18
F-ratio	NO2	0.900		ND		ND		ND
	NO3-	2.522		3.181		2.837		4.145
	NH4+	3.182		ND		ND		ND
	N-min	3.457		3.181		2.837		4.145

Figures are means of four replicates

F Ratio = Ratio of sum of squares ( treatment ) / sum of squares (error)

- LOQ NH4+: 0.26 mg/kg

- LOQ NO3-: 1.04 mg/kg

- LOQ NO2: 0.10 mg/kg

ND = not determined

*) significant (at p = 0.05)

N-min = Total mineralised nitrogen (i.e. total of ammonium + nitrate + nitrite (if present)

 

Table 4: Mean rate of Nitrate formation (mg NO3-/kg soil/dav) in a silty sand soil treated with the test substance’ metabolites

Treatment	mg N/kg soil/day
	0-7 Days	7-14 Days	14-28 Days	Overall rate (7-28 Days)
Control	0.45	1.65	1.38	1.47
Low rate	0.24 (-46.67)	1.66 (+0.61)	1.33 (-3.62)	1.44 (-2.04)
High rate	0.60 (+33.33)	1.79 (+8.48)	1.53 (+10.87)	1.62 (10.20)

( ) = % Variation from control treatment

Validity criteria fulfilled:

yes

Conclusions:

The metabolites, were applied at two rates, a low rate, equivalent to 0.6 mg/kg, 0.3 mg/kg and 0.4 mg/kg for M4, M5 and M9, respectively, or a higher rate, equivalent to 4.8mg/kg M4, 2.8 mg/kg M5 and 3.3mg/kg M9, respectively. Throughout the 28-day test duration, treatment of the soils with the metabolites did not result in ecologically significant effects at either treatment rate. Deviations from the control were clearlv below the trigger value of ±25% during the course of the study for the lower and higher rate treatment concentrations. Therefore, it was concluded that the metabolites of the parent substance do not affect the microorganisms in soil at high application rates.

Executive summary:

The influence of the metabolites of the parent substance (M4, M5 and M9) soil microflora was determined using short-term respiration experiments and by monitoring nitrogen transformation in accordance with OECD TG 216 and 217 and in compliance with GLP criteria. A mixture of relevant metabolites in distilled water was applied to a low organic matter silty sand soil, at a concentration equivalent to 0.6 mg/kg M4, 0.3 mg/kg M5 and 0.4 mg/kg M9. The test was also conducted at a higher rate, equivalent to 4.8 mg/kg M4, 2.8 mg/kg M5 and 3.3mg/kg M9 (assuming 100% distribution in soil to a depth of 5cm with a soil bulk density of 1.5 g/cm 3). The effects of the metabolites on microbial respiration were investigated using short-term respiration experiments conducted after 0, 7, 15 and 28 days. On each occasion, aliquots of soil were amended with a non-limiting quantity of glucose and carbon dioxide evolution measured over the subsequent 24-hour period using an infra-red gas analyser. Values for substrate induced respiration rate were converted to soil microbial respiration rates (mg CO2/kg/h) for data presentation. The effects on nitrogen transformation, ammonification and nitrification were investigated by determining ammonium-N, nitrate-N and nitrite-N concentrations in soil amended with ground Lucerne grass. Aliquots of soil were extracted with 2M KCl within 3 hours of treatment and after 0. 7, 14 and 28 days. The concentrations of inorganic nitrogen species in the extracts were determined colourimetrically. The rates of formation of nitrate (mg N/kg soil/day) were calculated for each treatment. Treatment of the soils with the metabolites did not result in ecologically significant effects at either treatment rate. Deviations from the control were clearly below the trigger value of ± 25% during the course of the study for the lower and higher rate treatment concentrations. 

Description of key information

- Test formulation: < 25% effect on C and N mineralization during 28 days at 12 mg/kg both in loamy sand and clay sand soil, OECD TG 216/217, McMurray 2002

- Metabolites: < 25% effect on C and N mineralization during 28 days at 2.8 - 4.8 mg/kg depending on metabolite, OECD TG 216/217, McMurray 2002

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

12 mg/kg soil dw

Additional information

Test formulation

The influence of the test formulation, a SC formulation of the test substance, on soil microflora was determined using short term respiration experiments and by monitoring nitrogen transformation in accordance with OECD TG 216 and 217. In addition, the study was performed using two soils, as described in the Dutch Standard NEN 5795, in order to provide for a wide applicability of the results obtained. A dispersion of The test formulation in distilled water was applied to two soils, one a high organic matter loamy sand soil and the other a low organic matter clayey sand soil at a nominal application rate of 8 mg/kg The test formulation. The test was also conducted at 12 mg/kg. The treatment rates were based on predicted environmental concentration calculations, taking into account the short half-life of the test substance. The effects of The test formulation on microbial respiration were investigated using short-term respiration experiments conducted after 0, 7, 14 and 28 days. On each occasion, aliquots of soil were amended with a non-limiting quantity of glucose and carbon dioxide evolution measured over the subsequent 24 hour period using an infra-red gas analyser. Values for substrate induced respiration rate were converted to soil microbial respiration (mg CO2/h/kg soil) for data presentation. The effects on nitrogen transformation, ammonification and nitrification were investigated by determining ammonium-N, nitrate-N and nitrite-N concentrations in soil amended with ground lucerne grass. Aliquots of soil were extracted with 2M KCl after 0, 7, 14, and 28 days and after 0, 7, 14, 28, 42, 56, 70, 84 and 98 days. The concentrations of inorganic nitrogen species in the extracts were determined colour metrically

When applied to a high organic matter loamy sand soil at 8 mg/kg and at 12 mg/kg, the test formulation did not give rise to any statistically significant deviations, in excess of ±25% during the course of the study. After 28 days incubation all deviations from control values were within ± 6%. The test formulation, when applied to a low organic matter clayey sand soil at 8 mg/kg and at 12 mg/kg, did not give rise to any statistically significant deviations, in excess of ±25% during the course of the study. After 28 days incubation all deviations from control values were within ±17%.When applied to a high organic matter loamy sand soil at 8 mg/kg and at 12 mg/kg, The test formulation did not cause any statistically significant long term effects on nitrogen transformation processes based on assessment of nitrate concentrations. After 28 days incubation the concentrations of nitrate measured in each treatment rate were found to be within ± 6% of the control values. The overall rate of formation of nitrate did not result in a statistically significant difference exceeding ± 25% variation from control values. 

The test formulation, when applied to a low organic matter clayey sand soil at 8 mg/kg and at 12 mg/kg had a transitory effect on nitrate concentrations. At the 8 mg/kg treatment rate this effect could be observed up to and including day 14. At day 28 and for the remainder of the study no statistically significant deviations in excess of ± 25% were observed at this treatment rate. This stimulatory effect was observed up until and including day 84 in the 12 mg/kg treatment rate. After 98 days incubation, no statistically significant deviations in excess of ± 25% were observed for nitrate concentrations. The overall rate of nitrate formation did not result in any statistically significant deviations. At the 8 mg/kg treatment rate a percentage deviation of 0.00% was observed. At the 12 mg/kg treatment rate a percentage deviation of 29.72% was observed.

The data presented in this report concludes that the test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and low organic matter clayey sand soil would not adversely affect soil microbial respiration. The test formulation when applied at 8 mg/kg and 12 mg/kg to a high organic matter loamy sand soil and at 8 mg/kg to a low organic matter clayey sand soil would not adversely affect soil microbial nitrogen transformation processes. Therefore, the 28-d NOEC was determined to be ≥ 12 mg/kg.

Metabolites

The influence of the metabolites of the parent substance (M1, M2 and M3) soil microflora was determined using short-term respiration experiments and by monitoring nitrogen transformation in accordance with OECD TG 216 and 217 and in compliance with GLP criteria. A mixture of relevant metabolites in distilled water was applied to a low organic matter silty sand soil, at a concentration equivalent to 0.6 mg/kg M1, 0.3 mg/kg M2 and 0.4 mg/kg M3. The test was also conducted at a higher rate, equivalent to 4.8 mg/kg M1, 2.8 mg/kg M2 and 3.3 mg/kg M3 (assuming 100% distribution in soil to a depth of 5cm with a soil bulk density of 1.5 g/cm 3). The effects of the metabolites on microbial respiration were investigated using short-term respiration experiments conducted after 0, 7, 15 and 28 days. On each occasion, aliquots of soil were amended with a non-limiting quantity of glucose and carbon dioxide evolution measured over the subsequent 24-hour period using an infra-red gas analyser. Values for substrate induced respiration rate were converted to soil microbial respiration rates (mg CO2/kg/h) for data presentation. The effects on nitrogen transformation, ammonification and nitrification were investigated by determining ammonium-N, nitrate-N and nitrite-N concentrations in soil amended with ground Lucerne grass. Aliquots of soil were extracted with 2M KCl within 3 hours of treatment and after 0. 7, 14 and 28 days. The concentrations of inorganic nitrogen species in the extracts were determined colourimetrically. The rates of formation of nitrate (mg N/kg soil/day) were calculated for each treatment. Treatment of the soils with the metabolites did not result in ecologically significant effects at either treatment rate. Deviations from the control were clearly below the trigger value of ± 25% during the course of the study for the lower and higher rate treatment concentrations.