2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

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:

30 June 2011 to 5 August 2011

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

A stock solution was prepared by dispersing/suspending 10 mg test material in 100 mL deionised water.
Appropriate amounts of stock solution were mixed to the soil using a laboratory mixer; in the course of the procedure the soil was ventilated and moistened with an adequate amount of deionised water. The soil water contents were adjusted to ca 46 - 47 % of the water holding capacity (WHCmax).

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 - 22 °C

Moisture:

45 - 48 % of its maximum water holding capacity

Details on test conditions:

TEST SYSTEM
- Test container: disposable plastic boxes; each box contained different soil masses for the two tests.
Soil respiration test: 750 - 1000 g soil (dry weight), box size ca. 1 L (0.12 x 0.165 x 0.065 m) filled up to 6 cm.
Nitrogen turnover test: 250 - 500 g (dry weight), box size ca. 0.5 L (0.1 x 0.1 x 0.065 m), filled up to 6 cm.
The soil was loosely filled into the boxes, which were covered by perforated lids to enable a slight, but sufficient air exchange
- No. of replicates per concentration: 3 units per treatment group
- No. of replicates per control: 3 units per treatment group

SOIL INCUBATION
- Method: bulk / series of individual sub-samples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Rossdorf, Germany (8° 44' 38.70" E, 49° 51' 59.59" N)
- Vegetation cover: fallow grassland
- Treatments with pesticides or fertilisers: no pesticide application for at least 4 years and no organic or mineral fertiliser for at least 4 years
- Depth of sampling: 0.05 - 0.2 m
- Soil texture
- % sand: 57.1
- % silt: 34.6
- % clay: 8.3
- Soil texture: silty loamy sand
- pH: 6.9
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 13.265
- Maximum water holding capacity (in % dry weight): 39.0
- Cation exchange capacity (mmol Ba/kg dry weight): 61
- Pre-treatment of soil: soil was air dried and sieved (2 mm mesh) at room temperature
- Storage (condition, duration): 20 days pre-incubation at 20 ± 2 °C
- Initial microbial biomass as % of total organic C: 2.57

DETAILS OF PRE-INCUBATION OF SOIL
Soil was maintained at 20 ± 2 °C, with appropriate ventilation and the soil was moistened periodically. The soil dry matter prior to the start of the test was ca. 86.2 %. The soil was stored in plastic containers with a sufficient headspace above the soil to allow gas exchange and the moisture was regulated by spraying deionised water on the surface.

EFFECT PARAMETERS MEASURED
- Determination of soil respiration in soil after addition of glucose.
Comparison of test material treated soil with a non-treated soil. Three replicates per treatment and concentration. A BSB-Sensomat System® was used to determine the oxygen consumption and CO₂-production over a period of up to 24 hours at different sampling intervals
- Determination of nitrogen-transformation (ammonium-, nitrite-, and nitrate-nitrogen levels) in soil enriched with lucerne meal (concentration in soil 0.5 %). Comparison of test material treated soil with a non-treated soil. Three replicates per treatment and concentration. NH₄-, NO₂-, and NO₃-nitrogen formed from the nitrification process were determined by means of Dionex ion chromatography system (DX-120 IC or ICS 1000, AS/AS 50 autosampler, ECD and UVD 340S photometer).
Sampling scheme: 0, 7, 14 and 28 days after treatment (soil respiration and soil nitrogen transformation tests). Sub-samples were withdrawn from the soil bulk batches and subjected to analysis.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

0 (control), 0.34, 1.69 mg/kg soil dw

Reference substance (positive control):

yes

Remarks:

sodium chloride

Key result

Duration:

28 d

Remarks on result:

other: the test material had no impact on respiration activity, soil nitrate content and soil nitrate formation rate of soil microflora when applied up to 1.69 mg/kg soil dry weight.

Details on results:

SOIL RESPIRATION RATES
The soil respiration rates were clearly within the trigger value of ± 25 % set by OECD guideline 217 throughout the experiment. On day 28 the values differed by 2.87 and 0.00 % from the control for the low and high dose rate, respectively. There were no statistically significant differences between control and test material treated groups within the experiment.

SOIL NITRATE CONTENT
No adverse effects of test material on nitrogen transformation in soil could be observed in both test material concentrations (0.34 mg/kg dry soil and 1.69 mg/kg dry soil) after 28 days. Only slight deviations from the control of -7.90 % (application rate 0.34 mg/soil dry weight) and -1.62 % (application rate 1.69 mg/soil dry weight) were measured at the end of the 28-day incubation period.

NITRATE FORMATION RATE
The soil nitrate formation rates were calculated on an incremental basis (i.e. between successive sampling dates). The difference in the soil nitrate formation rate between the control and both test concentrations was never higher than 25 % in the experiment. In the last interval between days 14 and 28, the deviations from control were -9.01 and -5.41 % for the lower and higher test concentration of test material. Thus, the difference in the soil nitrate formation rates between the control and both test material treatments was clearly below the OECD guideline 216 trigger value of 25 % at the 14 to 28 day interval. These deviations were statistically significant.

SOIL MINERAL NITROGEN CONTENT
The differences between the mineral nitrogen content of test material treated soil at both test concentrations and the control soil were also clearly below the 25 % trigger value at day 28 (required only by the EPPO and SETAC guidelines). At day 28 the differences were -7.79 and -2.60 %, respectively.

Results with reference substance (positive control):

The inhibition of soil respiration and nitrogen transformation by sodium chloride at a concentration of 16 g/kg soil dw is determined at least once a year as a means of assuring that the laboratory test conditions are adequate and have not changed significantly. Sodium chloride was applied to the soil at a concentration of 16 g/kg dry weight.
The results of the most recent study are as follows (reported as deviation from control on day 28 and day 99):
- Soil respiration rates: -68.14 %, -64.54 %
- Soil nitrate content: -96.47 %, -97.76 %
- Soil nitrate formation rate: -104.32 %, -100.00 %

All differences from control were statistically significant. The reference material was found to have a retarding or stimulating effect of more than ± 25 % compared to control at days 28 and 99 after application.

Reported statistics and error estimates:

Calculation of mean values per treatment, standard deviation and coefficient of variation. Normality and homogeneity of variances were tested using the R/S-Test (α = 0.05) and Cochran's test (α = 0.05), respectively, and pair-wise comparisons of treated and control values according to Student's t-test + Welch t-test (α = 0.05) were performed.

Table 1: Soil Respiration Test: Soil Respiration Rates in mg CO₂/kg soil dry weight/h (Mean Values)

Day	Control	Test material (mg/kg soil dw)
		0.34	1.69
0	7.341	7.474	7.793
7	9.850	8.608	8.696
14	7.371	6.976	6.893
28	7.073	7.276	7.073

Table 2: Nitrogen Transformation Test: Effects of Test Material on Soil Nitrogen Content in mg/kg soil dry weight (Mean Values)

Day	Control	Test material (mg/kg soil dw)
		0.34	1.69
	NH₄-N
0	4.514	3.095*	1.847*
7	1.768	1.441	1.344*
14	1.162	1.076	0.949*
28	1.501	1.351	1.203*
Day	NO₂-N
0	0.730	0.730	0.730
7	0.730	0.730	0.730
14	0.730	0.730	0.730
28	0.730	0.730	0.730
Day	NO₃-N
0	10.617	10.008*	10.496
7	2.022	2.223	2.369
14	8.515	7.986	8.964
28	24.109	22.205	23.719
Day	Mineral N
0	15.861	13.833*	13.072*
7	4.519	4.395	4.443
14	10.407	9.792	10.643
28	26.340	24.287	25.654

 *Significantly different from the control according to student t-test/Welch t-test, two sided, α = 0.05

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, it is concluded that the test material had no impact on respiration activity, soil nitrate content and soil nitrate formation rate of soil microflora when applied up to 1.69 mg/kg soil dry weight. It can be concluded that the test material will not have any long term influence on soil micro-organisms.

Executive summary:

The toxicity of the test material was investigated to soil microorganisms is a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 216 and OECD 217.

More specifically, in a 28 day study, the microbial respiration and nitrogen transformation rates were measured in a mid loamy sand soil, treated with test material at nominal test rates of 0 (control), 0.34 mg and 1.69 mg/kg soil.

At day 28, the respiration rates in soil treated with test material at concentrations of 0.34 mg and 1.69 mg test material per kg soil dry weight, were 2.87 and 0.00 % respectively, from the control mean. The results were below the 25 % criterion for effect as stated in test guideline OECD 217. At day 28, the nitrate transformation rates in soil treated with test material at concentrations of at 0.34 mg and 1.69 mg test material per kg soil dry weight, were -9.01 and -5.41 % respectively, from the control mean. The results were below the 25 % criterion for effect as stated in test guideline OECD 216.

Based on these results, the test material is not expected to cause any long-term toxicity to soil microbial processes.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2001 to 22 February 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

Principles of method if other than guideline:

During the study the test material was to be applied to a low organic matter silty sand soil at the field rate and 5 times that rate, equivalent to 0.168 mg a.s./kg and 0.84 mg a.s./kg, respectively. However a 1:10 dilution step was overlooked during sample preparation, this resulted in application rates of 1.68 mg a.s./kg (equivalent to 1200 g a.s./ha) for the lower concentration and 8.4 mg a.s./kg (equivalent to 6000 g a.s./ha) for the higher concentration.

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 95.4%

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

Application levels were achieved by preparing the following stock solutions of the test material in acetone:
- Stock solution - Lower rate: 168 mg/litre
- Stock solution - Higher rate: 840 mg/litre
Aliquots of the working stock dilution were applied to aliquots of soil at the rate of 10 mL per kg soil (dry weight).

Replicate batches of conditioned soil were treated with the test material by even distribution of quartz sand (containing the correct amount of test material) over the soil surface of each replicate, followed by thorough mixing. The control replicates were treated in a similar manner with quartz sand alone. All replicates were adjusted to 20.32 % w/w moisture content. All treatments were then covered with plastic lids containing eight small holes to allow gas exchange and were then incubated under aerobic conditions at 20 ± 2 °C.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

40 % WHC (20.32 % moisture)

Details on test conditions:

TEST SYSTEM
- Test container: Samples with dry weight equivalent to 1000 g were weighed into plastic boxes (18 x 18 x 9 cm) for respiration and samples with dry weight equivalent to 100 g were weighed into sample pots (7.5 cm diameter x 8 cm depth) for nitrogen mineralisation. The respiration sample containers were covered with plastic lids, containing 8 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.
- Treatment rate replication: 4 x 1000 g or 66 x 100 g dry weight equivalent soil replicates per treatment rate for respiration determination and nitrogen transformations, respectively.

SOIL INCUBATION
Following addition of soil to test containers the soil was incubated at a temperature of 20 ± 2 °C for 22 days prior to the start of the study.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil used in this study was obtained from Agronomy Enterprise
- Vegetation cover: grassland site
- Treatments with pesticides or fertilisers: No pesticide or fertiliser application within the previous 12 months
- Soil texture class: silty sand
- pH (in water): 6.5
- Maximum water holding capacity (% w/w soil): 50.8
- Cation exchange capacity (mEq/100 g): 8.4
- Pre-treatment of soil: air dried sufficiently to allow sieving through a 2 mm sieve
- Storage (condition, duration): 2 - 6 °C for 66 - 71 days at a moisture content of 13.15 %
- Organic carbon (%): 1.1
The soil microbial biomass at the start of the study was 0.037 g C/100 g soil. This represents 3.36 % of the total organic carbon; therefore, the soil was acceptable for use in this study.

EFFECT PARAMETERS MEASURED
The effects of the test material 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 12-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).
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 2 M KCl within 3 hours of treatment and after 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 rate.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

0 (control), 1.68, 8.4 mg a.s./kg

Reference substance (positive control):

yes

Remarks:

Dinoseb acetate

Key result

Duration:

28 d

Remarks on result:

other: the test material, when applied at the lower rate, equivalent to 1200 g a.s./ha and at a higher rate, equivalent to 6000 g a.s./ha, does not adversely affect soil microbial respiration or nitrogen transformation processes.

Details on results:

When applied to a low organic matter silty sand soil at the lower rate (equivalent to 1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 % during the course of the study. The control replicates met the ± 15 % variation criteria.
When applied to a low organic matter silty sand soil at the lower rate (equivalent to 1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 %, with regard to nitrogen transformation processes based on assessment of nitrate formation rates. After 28 days of incubation, the concentration of nitrate measured was found to be within ± 5 % of the control values. The rate of nitrate formation between 7 and 28 days displayed a percentage deviation not exceeding ± 25 % variation from control values. The control replicates met the ± 15 % variation criteria.

Results with reference substance (positive control):

The reference material (Dinoseb acetate) produced significant effects 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 treatment rates was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment rate 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 treatment rates was observed, a suitable mean comparison test was performed to determine statistical significance of each treatment rate at the 5 % level. Nitrate formation rates were calculated for each treatment rate, 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 rate at day 7, from the values after 28 days. The resulting concentrations were converted into rates and the data analysed as described 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 1: Effect of the Test Material on Mean Microbial Respiration Rate

Treatment	mg CO₂/h/kg soil
	Day 0	Day 7	Day 14	Day 28
Control	16.63	16.31	14.70	13.83
Lower rate	16.83	16.91	15.39	14.44
Higher rate	16.33	15.91	14.78	13.47
F ratio	0.638	5.575	2.909	2.640

Table 2: Effect of the Test Material on Mean NH₄+N, NO₃-N, NO₂-N and Total Mineral Nitrogen (N-min) Concentrations (Mean Values)

Treatment		mg N/kg soil
		Day 0	Day 7	Day 14	Day 28
Control	NO₂-	<0.10	<0.10	<0.10	<0.10
	NO₃-	27.35	26.22	33.70	51.93
	NH₄+	5.55	<0.52	<0.52	<0.52
	N-min	32.90	26.22	33.70	51.93
Lower rate	NO₂-	<0.10	<0.10	<0.10	<0.10
	NO₃-	30.88*	25.10	32.67	54.38
	NH₄+	6.50*	1.09	<0.52	<0.52
	N-min	37.38*	26.19	32.67	54.38
Higher rate	NO₂-	<0.10	<0.10	<0.10	<0.10
	NO₃-	31.43*	27.22	34.74	52.80
	NH₄+	5.28	0.85	<0.52	<0.52
	N-min	36.71*	28.07	34.74	52.80
F-Ratio	NO₂-	ND	ND	ND	ND
	NO₃-	20.243	1.070	0.720	0.424
	NH₄+	39.424	ND	ND	ND
	N-min	19.774	0.786	0.720	0.424

* % variance from the control significant at p = 0.05

ND - Not determined

Table 3: Mean Rate of Nitrate Formation (mg NO₃-/kg soil/day)

Treatment rate	mg N/kg soil
	0-7 days	7-14 days	14-28 days	Overall rate (7-28 days)
Control	-0.16	1.07	1.30	1.23
Lower rate	-0.82	1.08	1.55	1.39
Higher rate	-0.59	1.07	1.29	1.22

Validity criteria fulfilled:

yes

Conclusions:

It is concluded that the test material, when applied at the lower rate, equivalent to 1200 g a.s./ha and at a higher rate, equivalent to 6000 g a.s./ha, does not adversely affect soil microbial respiration or nitrogen transformation processes.

Executive summary:

The toxicity of the test material to soil microorganisms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 216 and OECD 217.

During the study the test material was to be applied to a low organic matter silty sand soil at the field rate and 5 times that rate, equivalent to 0.168 mg a.s./kg and 0.84 mg a.s./kg, respectively. However a 1:10 dilution step was overlooked during sample preparation, this resulted in application rates of 1.68 mg a.s./kg (equivalent to 1200 g a.s./ha) for the lower concentration and 8.4 mg a.s./kg (equivalent to 6000 g a.s./ha) for the higher concentration.

The effects of the test material 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 12-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 2 M KCl within 3 hours of treatment and after 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 rate.

The soil microbial biomass for the silty sand soil at the start of the study was 0.037 g C/100 g soil. This represents 3.36 % of the total organic carbon; therefore, the soil was acceptable for use in this study.

When applied to a low organic matter silty sand soil at the lower rate (equivalent to1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 % during the course of the study. The control replicates met the ± 15 % variation criteria.

When applied to a low organic matter silty sand soil at the lower rate (equivalent to 1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 %, with regard to nitrogen transformation processes based on assessment of nitrate formation rates. After 28 days incubation, the concentration of nitrate measured was found to be within ± 5 % of the control values. The rate of nitrate formation between 7 and 28 days displayed a percentage deviation not exceeding ± 25 % variation from control values. The control replicates met the ± 15 % variation criteria.

In consideration of the study findings, it is concluded that the test material, when applied at the lower rate, equivalent to 1200 g a.s./ha and at a higher rate, equivalent to 6000 g a.s./ha, does not adversely affect soil microbial respiration or nitrogen transformation processes.

Description of key information

The test material does not adversely affect soil microbial respiration or nitrogen transformation processes, OECD 216 and OECD 217, Feil (2011) & McMurray (2002)

Additional information

Two studies investigating the toxicity of the substance to soil microorganisms are available. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both studies were subsequently assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

In the study reported by Feil (2011) the toxicity of the test material was investigated to soil microorganisms is a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 216 and OECD 217.

More specifically, in a 28 day study, the microbial respiration and nitrogen transformation rates were measured in a mid loamy sand soil, treated with test material at nominal test rates of 0 (control), 0.34 mg and 1.69 mg/kg soil.

At day 28, the respiration rates in soil treated with test material at concentrations of 0.34 mg and 1.69 mg test material per kg soil dry weight, were 2.87 and 0.00 % respectively, from the control mean. The results were below the 25 % criterion for effect as stated in test guideline OECD 217. At day 28, the nitrate transformation rates in soil treated with test material at concentrations of at 0.34 mg and 1.69 mg test material per kg soil dry weight, were -9.01 and -5.41 % respectively, from the control mean. The results were below the 25 % criterion for effect as stated in test guideline OECD 216.

Based on these results, the test material is not expected to cause any long-term toxicity to soil microbial processes.

In the study reported by McMurray (2002) the toxicity of the test material to soil microorganisms was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 216 and OECD 217.

During the study the test material was to be applied to a low organic matter silty sand soil at the field rate and 5 times that rate, equivalent to 0.168 mg a.s./kg and 0.84 mg a.s./kg, respectively. However a 1:10 dilution step was overlooked during sample preparation, this resulted in application rates of 1.68 mg a.s./kg (equivalent to 1200 g a.s./ha) for the lower concentration and 8.4 mg a.s./kg (equivalent to 6000 g a.s./ha) for the higher concentration.

The effects of the test material 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 12-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 2 M KCl within 3 hours of treatment and after 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 rate.

The soil microbial biomass for the silty sand soil at the start of the study was 0.037 g C/100 g soil. This represents 3.36 % of the total organic carbon; therefore, the soil was acceptable for use in this study.

When applied to a low organic matter silty sand soil at the lower rate (equivalent to1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 % during the course of the study. The control replicates met the ± 15 % variation criteria.

When applied to a low organic matter silty sand soil at the lower rate (equivalent to 1200 g a.s./ha) and at the higher rate (equivalent to 6000 g a.s./ha), the test material did not give rise to any statistically significant deviations, in excess of ± 25 %, with regard to nitrogen transformation processes based on assessment of nitrate formation rates. After 28 days incubation, the concentration of nitrate measured was found to be within ± 5 % of the control values. The rate of nitrate formation between 7 and 28 days displayed a percentage deviation not exceeding ± 25 % variation from control values. The control replicates met the ± 15 % variation criteria.

In consideration of the study findings, it is concluded that the test material, when applied at the lower rate, equivalent to 1200 g a.s./ha and at a higher rate, equivalent to 6000 g a.s./ha, does not adversely affect soil microbial respiration or nitrogen transformation processes.