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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
(The test duration was 7 days against OECD 216 TG recommendation of 28 days. In this study, NO2 (nitrite) analysis was done instead of nitrate and no information is available for nitrite formation in each replicate control samples.)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
yes
Remarks:
(The test duration was 9 days against OECD 217 TG recommendation of 28 days, control as well as treated soils should be mixed with glucose as per TG but it was not added and no information is available for CO2 released from each replicate control samples)
Principles of method if other than guideline:
The aim of this study was to study the short-term effects of aqueous C10-C13 sodium LAS on microbial parameters in Danish sandy agricultural soil that was incubated for up to 11 days. The microbial soil parameters were related to carbon and nitrogen transformation (i.e., ethylene degradation, basal respiration, and ammonium oxidation), endo- and exoenzymatic activity (i.e., dehydrogenase activity and β-glucosidase activity), anaerobic activity (i.e., iron reduction), microbial populations (i.e., cellulolytic bacteria, fungi, and actinomycetes), and a broad indicator of microbial biomass (i.e., total content of phospholipid fatty acids [PLFA]). The EC10, EC50 and NOEC values were calculated at the end of exposure period.
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
For LAS analyses, duplicate soil samples of each batch (except batch prepared for Basal respiration measurements) were collected at the start of the incubation period and frozen (all LAS levels). LAS was extracted with methanol, analyzed by high performance liquid chromatography, and quantified as the sum of the C10 to C13 homologues. In each series of analyses, recoveries (range, 73.4–99.8%; n = 8) for duplicate standard soils with LAS concentrations of approximately 10 mg/kg dw were used to correct the high-performance liquid chromatography results for the test soils.
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Coarse sand from agricultural field (Lundgaard, Denmark)
- Other: The soil was acclimated at 15°C for approximately four weeks and sieved (4 mm) before the experiments.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Aqueous dilutions of the LAS stock solution were freshly prepared for addition to the soil. Appropriate solutions (1.25 mL) were added dropwise to triplicate 250-mL flasks to give LAS contents of 0, 8, 22, 62, 174, and 488 mg/kg (LAS concentrations are reported on a soil dw basis) and a gravimetric soil water content of 16%. The further details on soil incubation are provided in “Details on test conditions” section.

VEHICLE: No
Test organisms (inoculum):
soil
Total exposure duration:
11 d
Remarks:
Ethylene degradation: 0.5 day; Ammonium oxidation, dehydrogenase activity, β-Glucosidase activity, cellulolytic microorganisms (bacteria, fungi and actinomycetes): 7 days; Iron reduction: 5 days; Basal soil respiration: 1–9 days; PLFA content: 11 days
Test temperature:
15°C
Moisture:
Soil water content: 16%
Details on test conditions:
TEST SYSTEM
- Testing facility: Danish Institute of Agricultural Sciences, Department of Crop Physiology and Soil Science, Tjele, Denmark
- Test container: 250 mL conical flasks (18 flasks were used)
- Amount of soil: Different for all assays, ranged from 60 mg dw soil to 1 Kg.
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: No vehicle control was used

SOIL INCUBATION
- Method: Assays of potential ammonium oxidation, potential dehydrogenase activity, β-glucosidase activity, cellulolytic microorganisms, and PLFA were done with subsamples from the same batches of LAS-amended soil. These batches represented 50 g dry weight of soil in each of 18 conical, 250-mL flasks. Appropriate solutions (1.25 mL) were added dropwise to triplicate flasks to give LAS contents of 0, 8, 22, 62, 174, and 488 mg/kg dw (LAS concentrations are reported on a soil dry weight basis) and a gravimetric soil water content of 16%. All soils were carefully mixed and incubated in the dark at 15°C. Duplicate soil samples (~26 g dw) for LAS analyses were frozen at the start of the incubation period (all LAS levels). Microbiological assays were done after incubation and LAS exposure for 7 days, except for PLFA (11 days). Assays of ethylene degradation, iron reduction, and basal soil respiration were done with separate batches of LAS amended soil as described below.

ETHYLENE DEGRADATION: Ethylene (C2H4) degradation was activated by incubation of test soil (~1 kg) for four weeks under a headspace atmosphere with 1000 mLC2H4 L-. Thereafter, six soil portions (~132 g dw) were amended with appropriate LAS solutions (3.3 mL) to final contents of 0, 8, 22, 62, 174, and 488 mg/kg dw (final water content, 16%). The samples were mixed (using a spatula) and equilibrated for 2 hours. Duplicate soil samples (~26 g dw) for LAS analyses were frozen at the start of the incubation period (all LAS levels). The rate of C2H4 degradation was calculated from the C2H4 depletion during the time interval from t0 to t1.

POTENTIAL AMMONIUM OXIDATION: Soil (~8.6 g dw) from each of the 18 soil incubations (described above under Method) was weighed into 250-mL conical flasks. Then, 100 mL of 0.5 mM (NH4)2SO4 in 1 mM K2HPO4 (pH 7.2) were added, followed by 1 mL of 1 M NaClO3 to inhibit NO2-oxidation. The flasks were incubated on a rotary shaker at 20°C in the dark. After 15 minutes (t0) and again 6 hours later (t1), 3 mL of the soil slurries were transferred to 10-mL test tubes with 3 ml of 4 M KCl to stop microbial activity. The samples were centrifuged (2000 g, 5 minutes), and 2 mL of filtered (0.7 µm) supernatant were transferred to a cuvette for colorimetric NO2- analysis by the diazotization method.

POTENTIAL DEHYDROGENASE ACTIVITY: Soil (~1.3 g dw) from each of the 18 soil incubations (described above under Method) was weighed into each of four 10-mL test tubes. Then, 0.75 mL of 0.2 M KH2PO4 (pH 7.5) with 0.1% yeast extract was added to each test tube, followed by 1 mL of 0.4% INT in deionized water. All tubes were briefly vortexed and incubated in the dark at 20°C. After 15 minutes (t0) and again 4 hours later (t1), two of the four test tubes were transferred to an ice bath and amended with 5 mL of ethanol, vortexed, centrifuged (2000 g, 5 minutes), and filtered (0.7 µm). The absorbance at 485 nm was measured and compared to a standard curve prepared from iodonitrotetrazolium formazan (INT-F) in ethanol, correcting for blanks.

β-GLUCOSIDASE ACTIVITY: From each of the 18 soil incubations (described above under Method), approximately 60 mg dry weight of soil were transferred to 2 mL of 50 mM TRIS buffer (pH 5.0).

IRON REDUCTION: Reduction of soil Fe(III) to Fe(II) was tested with soil samples (~26 g dry wt) transferred to test tubes with 15 mL of 1% (w/w) yeast extract. Five replicates were amended with appropriate LAS solutions (0.66 mL) to obtain contents of 0, 8, 22, 62, 174, and 488 mg/kg. For all LAS levels, two replicates were frozen for LAS analysis, whereas three replicates were stoppered and incubated at 15°C for 5 days. After incubation, 5 mL of 2 M KCl was added under a stream of N2 gas. The samples were briefly shaken, allowed to stand for 15 minutes, and centrifuged (1000 g, 2 minutes). The Fe(II) -containing supernatant (~8 mL) was withdrawn and filtered (0.7 µm). The first 3 mL of the filtrate were discarded, and then 0.5 mL of filtrate was rapidly transferred to 4.5 mL of 0.1% ferrozine in 50 mM N-2-hydroxyethylpiperazine-N’-2-ethane-sulfonic acid buffer (pH 7.0)]. The absorbance at 562 nm was measured and compared to a standard curve prepared from (NH4)2Fe(SO4)2 X 6H2O. The Fe(III) reduction rate was calculated from the accumulation of Fe(II) during the incubation period.

CELLULOLYTIC MICROORGANISMS: From each of the 18 soil incubations (see Experimental), a soil sample (~8.6 g dw) was transferred to 100 ml of deionized water and homogenized for 15 s (model 400 stomacher; Seward, London, UK). Serial dilutions were prepared from each suspension, and plate counts of cellulose-utilizing bacteria, fungi, and actinomycetes were done in triplicate by use of cellulose–Congo red agar.

BASAL RESPIRATION: Duplicate soil samples (~30 g dw) were amended with appropriate LAS solutions (1.5 mL) to final contents of 0, 0.8, 8, 79, and 793 mg/kg (final gravimetric water content, 18.5%). The samples were mixed and incubated at 15°C in sealed, 300-mL bottles with septa for gas sampling. Gas samples (1 mL) were withdrawn after incubation for 1, 2, 4, 5, and 9 days, and CO2 was quantified by GC with thermal conductivity detection.

PHOSPHOLIPID FATTY ACIDS: Two soil incubations at each LAS level (described above under Method) were subsampled (~1.7 g dw soil) for PLFA analysis after 11 days.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical location: Soil from the plough layer was sampled at an agricultural field (Lundgaard, Denmark) in August 1997. This soil type represents approximately 25% of the cultivated area in Denmark.
- History of site: The soil had not previously been treated with sewage sludge
- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: Soil had not been sprayed with pesticides the last two years.
- Accidental contamination: Not specified

- Depth of sampling: Plough layer
- Soil texture: Coarse sand soil
- Coarse sand, 200–2000 µm (%): 66.9
- Fine sand, 63–200 µm (%): 15.8
- Coarse silt, 20–63 µm (%): 3.3
- Fine silt, 2–20 µm (%): 5.3
- Clay, <2 µm (%): 6.2
- Iron oxy-hydroxides (dithionite) (ppm): 3800
- Aluminium oxy-hydroxides (dithionite) (ppm): 1410
- Humus (%): 2.7
- Density (g/cm3 dry soil): 1.263
- Soil taxonomic classification: Not specified
- Soil classification system: Not specified
- pH (in water): 5.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Not specified
- Maximum water holding capacity (in % dry weight): Not specified
- Cation exchange capacity (mmol/kg): 2.89 mEq/100 g (Ca2+), 0.16 mEq/100 g (K+), 0.12 mEq/100 g (Mg 2+), 0.6 mEq/100 g (Na+), 3.23 mEq/100 g (total)
- Pretreatment of soil: No
- Storage (condition, duration): After sampling, the soil was stored at 2°C in the field-moist condition (gravimetric water content, 13.5%) for approximately two months.
- Total carbon content: 1.5%

DETAILS OF PREINCUBATION OF SOIL (if any): The soil was acclimated at 15°C for approximately four weeks and sieved (4 mm) before the experiments.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Ethylene degradation, ammonium oxidation, dehydrogenase activity, β-Glucosidase activity, iron reduction, cellulolytic bacteria, cellulolytic fungi, cellulolytic actinomycetes, basal soil respiration and phospholipid fatty acid content.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY: No

Nominal and measured concentrations:
Nominal concentrations for all parameters (except basal soil respiration test): 0, 8, 22, 62, 174 and 488 mg/kg dw
Measured concentrations: <1, 7 ± 1, 21 ± 2, 59 ± 6, 149 ± 21 and 407 ± 52 mg/kg dw
Recovery was in range of 84 to 95% of the nominal concentrations.

Nominal concentrations for basal soil respiration (CO2 evolution): 0, 0.8, 8, 79 and 793 mg/kg dw
Reference substance (positive control):
no
Key result
Duration:
0.5 d
Dose descriptor:
EC10
Effect conc.:
9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: carbon formation rate (ethylene degradation)
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrite formation rate (ammonium oxidation)
Remarks on result:
other: 95% CL: 2 - 8 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
22 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dehydrogenase activity
Remarks on result:
other: 95% CL: 6 - 47 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
47 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: β-Glucosidase activity
Remarks on result:
other: 95% CL: 24 - 63 mg/kg soil dw
Key result
Duration:
5 d
Dose descriptor:
EC10
Effect conc.:
< 8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Iron reduction
Remarks on result:
other: 95% CL: 2 - 6 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Cellulolytic bacteria (microbial populations)
Remarks on result:
other: 95% CL: 0 -12 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Cellulolytic fungi (microbial populations)
Remarks on result:
other: 95% CL: 0 - 20 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Cellulolytic actinomycetes (microbial populations)
Remarks on result:
other: 95% CL: 0 - 19 mg/kg soil dw
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 793 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: carbon formation rate (basal soil respiration)
Key result
Duration:
11 d
Dose descriptor:
EC10
Effect conc.:
> 488 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total content of phospholipid fatty acids (broad indicator of microbial biomass)
Details on results:
EFFECTS ON SOIL MICROBIOLOGY

ETHYLENE DEGRADATION: The LAS had an inhibitory effect on soil C2H4 degradation, which almost ceased at the highest tested concentration. The inhibitory effect of LAS occurred rapidly after the initial LAS exposure i.e. 0.5 and 6 days of LAS exposure, and that no recovery occurred within 6 days.

POTENTIAL AMMONIUM OXIDATION: The inhibitory effect of LAS on the potential ammonium oxidation was significant at all the tested dose levels.

POTENTIAL DEHYDROGENASE ACTIVITY: Dehydrogenase activity was progressively inhibited at increasing LAS contents. However, the EC10 and EC50 values were relatively high (22 and 128 mg/kg, respectively) as compared to those for a range of other microbial parameters. Dehydrogenase enzymes are considered to be inactive in the extracellular form, and the potential dehydrogenase activity is, therefore, widely used as a measure of microbial biomass and activity. Dehydrogenase activity comprises the response of many groups of microorganisms. This is a likely reason for the lower effect of LAS toward dehydrogenase activity than toward processes dependent on individual species or groups of microorganisms.

IRON REDUCTION: Bacterial iron reduction was completely inhibited at 62 mg/kg. However, as opposed to the other microbial parameters tested, the LAS exposure during the iron-reduction test was done in a soil–water slurry, which may have increased the bioavailability (and toxicity) of LAS.

CELLULOLYTIC MICROORGANISMS: The three groups of cellulolytic microorganisms (bacteria, fungi and actinomycetes) showed a similar and sensitive response to LAS. Thus, the no-observed effect concentration and lowest-observed-effect concentration values for all three groups were 8 and 22 mg/kg, respectively. This sensitive response probably reflects that microbial cellulose degradation is a complicated process performed by specialized organisms. If LAS inhibit these microorganisms, no other p arts of the microbial community can start the degradation of cellulose.

β-GLUCOSIDASE ACTIVITY: The LAS inhibited β-glucosidase activity by only 25% at the highest tested concentration of 488 mg/kg. This moderate toxicity of LAS toward β-glucosidase activity was in accordance with the theory of LAS toxicity, which is generally ascribed to interactions with cell membranes and disruption of their proper functioning. Therefore, higher concentrations of LAS would be expected for inhibition of soil enzymatic processes than for inhibition of processes depending directly on the functional integrity of microbial cell membranes.

SOIL RESPIRATION: At 0.8 to 793 mg/kg, LAS did not inhibit basal soil respiration but, rather, caused a slight increase in CO2 production at the highest LAS contents. Such an increase could be caused by several mechanisms. For example, an increased CO2 production by LAS-resistant microorganisms may result from degradation of LAS or of microbial C from LAS-susceptible cells. In addition, the adsorption/desorption and surfactant properties of LAS may cause a mobilization of nutrients and degradable organic matter in the soil. Physiological mechanisms, such as stress-induced metabolism by LAS-susceptible cells, may also result in elevated respiration rates. For such reasons, a partial or complete compensation of inhibitory LAS effects may occur in terms of CO2 production, even though
a decrease in biomass or important changes in microbial community structure and stability have taken place.

PHOSPHOLIPID FATTY ACIDS: No effect of LAS on the total PLFA concentration, because the total soil PLFA concentration was approximately 50 nmol/g dry weight at all LAS concentrations. This lack of response was surprising considering the inhibitory effects of LAS on other indices of microbial biomass reported elsewhere, such as biomass C, adenosine triphosphate, and SIR. Hence, some decrease in microbial biomass (and total PLFA) was expected, at least at the higher LAS concentrations. It is not clear whether the present results represent a true indication of unchanged microbial biomass or if some methodological problems were involved (e.g., due to inhibition of phospholipases by LAS). If the PLFA content was truly unaffected by LAS, this could be explained by the fact that most bacteria were still present in the soil but were partly inactivated. In conclusion, however, the response of PLFA to LAS needs to be further investigated.
Reported statistics and error estimates:
Calculation of EC10 and EC50 values was done by a linear interpolation analysis (ICp), which was based on bootstrapping and included the 95% confidence limits for the estimates. The no-observed-effect and lowest-observed-effect concentrations were determined by Dunnett’s test using a SAS analysis of variance procedure.

Table 1: Effect concentrations (EC10, EC50, NOEC and LOEC) for linear alkylbenzene sulfonate (LAS) toward microbial parameters in agricultural soil. All data are presented as mg/kg soil dw (Elsgaard et al., 2001a)

Microbial parameter

Incubation
(in days)

EC10 value

95% CL for EC10 value

EC50 value

95% CL for EC50 value

NOEC

LOEC

Ethylene degradation

0.5

9

NAa

24

NA

NA

NA

Ammonium oxidation

7

<8

(2–8)

40

(24–76)

0

10

Dehydrogenase activity

7

22

(6–47)

128

(99–154)

22

62

β-Glucosidase activity

7

47

(24–63)

>488

NA

174

488

Iron reduction

5

<8

(2–6)

17

(15–18)

0

8

Cellulolytic bacteria

7

11

(0–12)

24

(15–40)

8

22

Cellulolytic fungi

7

<8

(0–20)

32

(11–76)

8

22

Cellulolytic actinomycetes

7

8

(0–19)

80

(0–206)

8

22

Basal soil respiration

1–9

>793

NA

>793

NA

>793

>793

PLFAbcontent

11

>488

NA

>488

NA

>488

>488

a NA: not available

b PLFA: phospholipid fatty acid

Validity criteria fulfilled:
yes
Conclusions:
In a short-term soil microbial toxicity study of aqueous C10-C13 sodium LAS at nominal concentrations of 0, 8, 22, 62, 174 and 488 mg/kg dw, there was adverse effect on ethylene degradation, potential ammonium oxidation, potential dehydrogenase activity, iron reduction, the populations of cellulolytic microorganisms (bacteria, fungi and actinomycetes) at one or more dose levels of LAS. The resultant EC10 values were in the range of less than 8 to 22 mg/kg dry weight. β-glucosidase activity, the basal soil respiration and the phospholipid fatty acid (PLFA) content were least effected by C10-C13 sodium LAS exposure. The extracellular β-glucosidase activity was rather insensitive to C10-C13 sodium LAS with EC10 value of 47 mg/kg dw whereas the basal soil respiration was not inhibited even at 793 mg/kg dry weight. The PLFA content showed no decrease even at 488 mg/kg dw.
Executive summary:

The aim of this study was to study short-term effects of aqueous C10-C13 sodium LAS on microbial parameters in a sandy agricultural soil that was incubated for up to 11 days.

 

The microbial soil parameters were related to carbon and nitrogen transformation (i.e., ethylene degradation, basal respiration, and ammonium oxidation), endo- and exoenzymatic activity (i.e., dehydrogenase activity and β-glucosidase activity), anaerobic activity (i.e., iron reduction), microbial populations (i.e., cellulolytic bacteria, fungi, and actinomycetes), and a broad indicator of microbial biomass (i.e., total content of phospholipid fatty acids [PLFA]). Agricultural soil samples were incubated with test substance at nominal concentrations of 0, 8, 22, 62, 174 and 488 mg/kg dw (measured concentrations: <1, 7 ± 1, 21 ± 2, 59 ± 6, 149 ± 21 and 407 ± 52 mg/kg dw) for all the microbial soil parameters (except basal soil respiration test) for a period of 0.5-11 days. The recovery was in range of 84 to 95% of the nominal concentrations. The test concentrations for basal soil respiration (CO2 evolution) were 0, 0.8, 8, 79 and 793 mg/kg dw.

 

The LAS had an inhibitory effect on soil ethylene degradation, which almost ceased at the highest tested concentration. The inhibitory effect of LAS on the potential ammonium oxidation was also significant at all the tested dose levels. The inhibition of dehydrogenase activity was progressively increased at increasing LAS contents. Bacterial iron reduction was completely inhibited at 62 mg/kg. The cellulolytic microorganisms (bacteria, fungi and actinomycetes) also showed an increase in inhibition of number of colony forming units in dose dependent manner and achieve significance at 22 mg/kg dw and above test concentrations.

 

Soil respiration was not inhibited at any of the test concentration but, rather, caused a slight increase in CO2 production at the highest LAS contents. No effect was observed for total phospholipid fatty acids concentration. The LAS inhibited β-glucosidase activity by only 25% at the highest tested concentration of 488 mg/kg.

 

The resultant EC10 values for ethylene degradation, potential ammonium oxidation, potential dehydrogenase activity, iron reduction, the populations of cellulolytic microorganisms (bacteria, fungi and actinomycetes) were in the range of less than 8 to 22 mg/kg dry weight. The extracellular β- glucosidase activity was rather insensitive to C10-C13 sodium LAS with EC10 value of 47 mg/kg dw whereas the basal soil respiration was not inhibited even at 793 mg/kg dry weight. The PLFA content showed no decrease even at 488 mg/kg dw.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
(Variation between nitrate concentrations of replicate control samples should be less than ± 15%. In this study, NO2 (nitrite) analysis was done instead of nitrate and no information is available for nitrite formation in each replicate control samples)
Principles of method if other than guideline:
The aim of this study was to study the short-term effects of aqueous C10-C13 sodium LAS and LAS-spiked sewage sludge on microbial parameters in Danish sandy agricultural soil that was incubated for 5 days to eight weeks. The evaluated microbial soil parameters were iron reduction, ammonium oxidation, dehydrogenase activity, arylsulfatase activity and microbial biomass C. The effects of aqueous LAS and LAS-spiked sewage sludge were compared and thereafter EC10, EC50 and NOEC values were calculated.
GLP compliance:
no
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Coarse sand from agricultural field (Lundgaard, Denmark) and sewage
sludge from wastewater treatment plant (Skaevinge, Denmark)

- Other: The soil was acclimated at 15°C for approximately four weeks and sieved (4 mm) before the experiments.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method for aqueous LAS solutions: For the experiments with aqueous LAS, seven triplicate soil incubations were amended with 14.32 g of appropriate LAS solutions to give the resulting LAS contents of 0, 3, 8, 22, 62, 174, and 488 mg/kg soil dw. The final gravimetric soil water content was 16%.

- Method for LAS-spiked sewage sludge: The sludge had a dry-matter content of 244 g/kg. Six sludge samples of 18.76 g (wet wt) were mixed with 50 mL of LAS dilutions containing 0, 1.58, 4.42, 12.36, 34.62, or 96.94 g/L stock solution. The samples were homogenized using a spatula and incubated at 5°C under an N2 atmosphere for 18 hours to allow the sludge–LAS mixture to equilibrate without oxic degradation of LAS. For the experiments with LAS-spiked sewage sludge, six triplicate soil incubations were amended with 15.34 g (wet wt) of the appropriate LAS-spiked sludge samples to give total LAS contents of 0, 8, 22, 62, 174, and 488 mg/kg soil dw; a sludge content of 2.86 g/kg dry weight; and a final gravimetric water content of 16%.

VEHICLE: No
Test organisms (inoculum):
soil
Total exposure duration:
8 wk
Remarks:
Iron reduction: 15 days; Ammonium oxidation, dehydrogenase activity, arylsulfatase activity and biomass C: 8 weeks
Test temperature:
15°C
Moisture:
Soil water content: 16%
Details on test conditions:
SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical location: Soil from the plough layer was sampled at an agricultural field (Lundgaard, Denmark) in June 1998.
- History of site: The soil had not previously been treated with sewage sludge
- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: Soil had not been sprayed with pesticides the last two years.
- Accidental contamination: Not specified
- Depth of sampling: Plough layer
- Soil texture: Coarse sand soil
- Coarse sand, 200–2000 µm (%): 66.9
- Fine sand, 63–200 µm (%): 15.8
- Coarse silt, 20–63 µm (%): 3.3
- Fine silt, 2–20 µm (%): 5.3
- Clay, <2 µm (%): 6.2
- Iron oxy-hydroxides (dithionite) (ppm): 3800
- Aluminium oxy-hydroxides (dithionite) (ppm): 1410
- Humus (%): 2.7
- Density (g/cm3 dry soil): 1.263
- Cation exchange capacity: 2.89 mEq/100 g (Ca2+), 0.16 mEq/100 g (K+), 0.12 mEq/100 g (Mg 2+),
0.6 mEq/100 g (Na+), 3.23 mEq/100 g (total)
- Soil taxonomic classification: Not specified
- Soil classification system: Not specified
- pH (in water): 5.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Not specified
- Maximum water holding capacity (in % dry weight): Not specified
- Pretreatment of soil: No
- Storage (condition, duration): After sampling, the soil was stored at 2°C in the field-moist condition
(gravimetric water content, 13.5%) for approximately two months.
- Total carbon content: 1.5%

SOURCE AND PROPERTIES OF SEWAGE SLUDGE
- Geographical location: Wastewater treatment plant at Skaevinge, Denmark.
- other: The sludge from the plant contained low amounts of xenobiotic compounds such as LAS, which ranged from less than 100 to approximately 600 mg/kg dw on two different sampling dates.

DETAILS OF PREINCUBATION OF SOIL (if any): The soil was acclimated at 15°C for approximately four weeks and sieved (4 mm) before the experiments.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Ammonium oxidation, dehydrogenase activity, iron reduction, arylsulfatase activity, and Microbial biomass C.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY: No
Nominal and measured concentrations:
Nominal concentrations: 0, 3, 8, 22, 62, 174 and 488 mg/kg soil dw

Measured concentrations for aqueous (aq) LAS study: <1, 3 ± 1, 6 ± 1, 16 ± 2, 51 ± 6, 141 ± 15 and
380 ± 25 mg/kg soil dw
Recovery was in range of 73 to 100% of the nominal concentrations.

Measured concentrations for LAS-spiked sewage sludge (ss) study: <1, NA, 6 ± 1, 16 ± 2, 44 ± 10,
118 ± 13 and 370 ± 18 mg/kg soil dw
Recovery was in range of 68 to 76% of the nominal concentrations.
Reference substance (positive control):
no
Key result
Duration:
15 d
Dose descriptor:
EC10
Effect conc.:
14 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Iron reduction
Remarks on result:
other: Aqueous LAS samples (without sewage sludge); 95% CL: 11–17 mg/kg soil dw
Key result
Duration:
15 d
Dose descriptor:
EC10
Effect conc.:
26 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Iron reduction
Remarks on result:
other: LAS with sewage sludge; 95% CL: 1-33 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
33 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrite formation rate (ammonium oxidation)
Remarks on result:
other: Aqueous LAS samples (without sewage sludge); 95% CL: 0-43 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
68 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrite formation rate (ammonium oxidation)
Remarks on result:
other: LAS with sewage sludge; 95% CL: 0-113 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
< 8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dehydrogenase activity
Remarks on result:
other: Aqueous LAS samples (without sewage sludge); 95% CL: 0–474 mg/kg soil dw
Key result
Duration:
8 d
Dose descriptor:
EC10
Effect conc.:
58 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dehydrogenase activity
Remarks on result:
other: LAS with sewage sludge; 95% CL: 0–693 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
75 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Arylsulfatase activity
Remarks on result:
other: Aqueous LAS samples (without sewage sludge); 95% CL: 0–90 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
> 488 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Arylsulfatase activity
Remarks on result:
other: LAS with sewage sludge
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass C
Remarks on result:
other: Aqueous LAS samples (without sewage sludge); 95% CL: 0–37 mg/kg soil dw
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
109 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass C
Remarks on result:
other: LAS with sewage sludge; 95% CL: 89–161 mg/kg soil dw
Details on results:
LAS CONCENTRATIONS: The nominal LAS levels, which were used for the calculation of effect concentrations, were adequately verified by the chemical analyses. The average recovery was 82% (n = 6) for soil samples with aqueous LAS and 73% (n = 5) for soil samples with LAS-spiked sludge. LAS degraded aerobically by more than 73% after two weeks for 8 to 62 mg/kg soil dw dose levels. No effect was observed from the addition of sludge on the LAS depletion after two weeks. Soil treated with aqueous LAS to 488 mg/kg soil dw showed only 15% LAS depletion. High LAS concentration may have inhibited microbial depletion or caused a prolonged lag phase to occur before LAS depletion. In soil samples incubated anaerobically for approximately two weeks (iron-reduction test, 15 d), LAS was more persistent than during the aerobic soil incubation.

IRON REDUCTION: In LAS control treatments at 0 mg/kg soil dw, the iron reduction in soil with or without sewage sludge was stimulated by 65 and 42%, respectively, during the 15-days incubation period. The level of iron reduction in the two soil treatments (with or without sludge) was similar at all three sampling times. This indicated sewage sludge was not an important source of iron-reducing bacteria to the soil - sludge system, but that these microorganisms were, rather, derived from the soil. After 5 days of incubation, there was a complete absence of iron reduction at concentrations greater than 62 mg/kg soil dw. After 10 and 15 days of incubation, iron reduction appeared at 62 mg/kg soil dw in both soil treatments and at 174 mg/kg soil dw in the sludge-amended soil. Therefore, LAS applied with sewage sludge was less toxic than aqueous LAS, and that the toxicity of LAS to iron reduction decreased during the incubation period.

POTENTIAL AMMONIUM OXIDATION: In control treatments at 0 mg/kg soil dw, the potential ammonium oxidation in soil with or without sludge showed an increase of 32 and 21%, respectively, during the eight-week incubation period. The sludge amendment stimulated the potential ammonium oxidation at all four sampling occasions by, on average, 43%. This indicated the presence of ammonium-oxidizing bacteria in the sludge. The inhibitory effect of LAS after one week of incubation was more pronounced in soil with aqueous LAS than with LAS-spiked sewage sludge. No ammonium oxidation was observed at 174 and 488 mg/kg soil dw levels without sewage sludge, however, ammonium oxidation was detected with sewage sludge samples at same concentrations. This effect of sludge application persisted throughout the incubation period of one, two, and four weeks but was less pronounced after eight weeks of incubation. After eight weeks of incubation, the potential ammonium oxidation occurred (albeit at low rates) at both the 174 and 488 mg/kg soil dw level.

POTENTIAL DEHYDROGENASE ACTIVITY: In control treatments at 0 mg/kg soil dw, the sludge amendment caused a general increase in the potential dehydrogenase activity at all sampling occasions. The average stimulation was 54%, as calculated from data obtained after two, four, and eight weeks of incubation. Data for the one-week exposure to aqueous LAS were obtained with soil samples that had been frozen prior to the assay. The dehydrogenase activity at all sampling occasions showed that the toxicity was higher for aqueous LAS than for LAS-spiked sewage sludge. However, there was partial or complete recovery of the LAS inhibition was seen after eight weeks of incubation.

ARYLSULFATASE ACTIVITY: In control treatments at 0 mg/kg soil dw, the level of arylsulfatase activity was similar in soil treatments with or without sewage sludge after incubation for four and eight weeks. After four and eight weeks of incubation with aqueous LAS, the arylsulfatase activity was inhibited only at the level of 488 mg/kg soil dw. However, no such inhibition was seen in LAS-spiked sewage sludge samples at any of the incubation periods.

MICROBIAL BIOMASS C: In control treatments (0 mg/kg soil dw), the microbial biomass C was approximately halved during the eight-week incubation period in soil treatments with or without sewage sludge. The inhibitory effect of LAS after one and eight weeks of incubation was less pronounced when LAS was added with sewage sludge than as an aqueous solution. However, the effects of aqueous on biomass C (without sewage sludge) LAS were slightly reduced by the longer incubation time.
Results with reference substance (positive control):
No reference substance was included in the study.
Reported statistics and error estimates:
Calculation of the EC10 and EC50 was done by the inhibition concentration (ICp) interpolation method. For one assay (biomass C in sludge-amended soil after one week of incubation), the EC10 and EC50 were estimated by a simple arithmetic interpolation, because insufficient data were available for the ICp procedure. No-observed-effect concentrations and lowest-observed-effect concentrations were determined by Dunnett’s test using an SAS analysis-of-variance procedure.

Table 1: Effect concentrations for aqueous linear alkylbenzene sulfonates (aq LAS) and sludge-applied LAS (LAS + sludge) toward microbial parameters in a sandy agricultural soil during incubation for 5 days to eight weeks (wk). All data are presented as mg/kg soil dw. (Elsgaard et al., 2001b)

Parameter Treatment Incubation EC10 value 95% CL for EC10 value EC50 value 95% CL for EC50 value NOEC LOEC
Iron reduction Aq LAS 5 days 3 (0–17) 17 (11–22) 3 8
LAS + sludge 5 days <8 (2–12) 30 (24–35) <8 8
Aq LAS 10 days 12 (10–13) 32 (30–34) 8 22
LAS + sludge 10 days 24 (0–32) 47 (40–52) 22 62
Aq LAS 15 days 14 (11–17) 39 (33–44) 8 22
LAS + sludge 15 days 26 (1–33) 55 (44–68) 22 62
Ammonium oxidation Aq LAS 1 wk 23 (0–32) 56 (48–64) 22 62
LAS + sludge 1 wk 89 (77–135) 230 (103–329) 62 174
Aq LAS 2 wk 18 (14–24) 62 (58–70) 8 22
LAS + sludge 2 wk 102 (91–118) 272 (244–294) 62 174
Aq LAS 4 wk 14 (6–25) 57 (49–65) 8 22
LAS + sludge 4 wk 121 (0–231) 303 (252–350) 174 488
Aq LAS 8 wk 33 (0–43) 102 (14–142) 62 174
LAS + sludge 8 wk 68 (0–113) 234 (137–320) 62 174
Dehydrogenase activity Aq LAS 1 wk 39 (30–57) 116 (84–201) 62 174
LAS + sludge 1 wk <22 (0–79) >488 NAe 62 174
Aq LAS 2 wk <8 (0–99) 335 (0–595) 62 174
LAS + sludge 2 wk <22 (0–71) >488 NA 22 62
Aq LAS 4 wk <8 (0–23) 71 (0–257) 8 22
LAS + sludge 4 wk 28 (0–84) >488 NA >488 >488
Aq LAS 8 wk <8 (0–474) 459 (0–515) >488 >488
LAS + sludge 8 wk 58 (0–693) >488 NA >488 >488
Arylsulfatase activity Aq LAS 4 wk 222 (203–228) 419 (289–444) 174 488
LAS + sludge 4 wk >488 NA >488 NA >488 >488
Aq LAS 8 wk 75 (0–90) 339 (319–360) 62 174
LAS + sludge 8 wk >488 NA >488 NA >488 >488
Biomass C Aq LAS 1 wk 13 (0–43) 48 (27–74) 22 62
LAS + sludge 1 wk 53 NA 487a NA NA NA
Aq LAS 8 wk 5 (0–37) 118 (28–159) 62 174
LAS + sludge 8 wk 109 (89–161) 361 (262–469) 174 488

NA: Not available

a Estimated by arithmetic interpolation, omitting an intermittent decrease in microbial biomass C.

Validity criteria fulfilled:
yes
Conclusions:
In a short-term soil microbial toxicity study of aqueous C10-C13 sodium LAS and LAS-spiked sewage sludge at nominal concentrations of 0, 3, 8, 22, 62, 174 and 488 mg/kg soil dw, presence of sewage sludge and prolonging (two to eight weeks) incubation time decreased the toxicity of LAS based on iron detection, potential ammonium oxidation, potential dehydrogenase activity, arylsulfatase activity and microbial biomass C. The EC10 values were <8 to 75 for aqueous LAS samples and 26 to >488 mg/kg soil dw for LAS-spiked sewage sludge samples.
Executive summary:

The aim of this study was to compare the short-term effects of aqueous C10-C13 sodium LAS and LAS-spiked sewage sludge on microbial parameters in a sandy agricultural soil that was incubated for 5 days to eight weeks.

The microbial soil parameters were iron reduction, ammonium oxidation, dehydrogenase activity, arylsulfatase activity and microbial biomass C. Agricultural soil samples were incubated with LAS or LAS spiked sewage sludge at nominal concentrations of 0, 3, 8, 22, 62, 174 and 488 mg/kg dw. The measured concentrations <1, 3 ± 1, 6 ± 1, 16 ± 2, 51 ± 6, 141 ± 15 and 380 ± 25 mg/kg dw soil for aqueous LAS study and <1, NA, 6 ± 1, 16 ± 2, 44 ± 10, 118 ± 13 and 370 ± 18 mg/kg dw soil for LAS-spiked sewage sludge study. The recovery was in range of 84 to 95% of the nominal concentrations for aqueous LAS study and 71 to 76% for LAS-spiked sewage sludge study.

After 5 days of incubation, there was a complete absence of iron reduction at concentrations greater than 62 mg/kg soil dw. After 10 and 15 days of incubation, iron reduction appeared at 62 mg/kg soil dw in both soil treatments and at 174 mg/kg soil dw in the sludge-amended soil. No ammonium oxidation was observed at 174 and 488 mg/kg soil dw levels without sewage sludge, however, ammonium oxidation was detected with sewage sludge samples at same concentrations. This effect of sludge application persisted throughout the incubation period of one, two, and four weeks but was less pronounced after eight weeks of incubation. The dehydrogenase activity at all sampling occasions showed that the toxicity was higher for aqueous LAS than for LAS-spiked sewage sludge. However, there was partial or complete recovery of the LAS inhibition was seen after eight weeks of incubation. After four and eight weeks of incubation with aqueous LAS, the arylsulfatase activity was inhibited only at the level of 488 mg/kg soil dw. However, no such inhibition was seen in LAS-spiked sewage sludge samples at any of the incubation periods. The inhibitory effect of LAS after one and eight weeks of incubation was less pronounced when LAS was added with sewage sludge than as an aqueous solution. The effects of aqueous on biomass C (without sewage sludge) LAS were slightly reduced by the longer incubation time.

The EC10 values were <8 to 75 mg/kg soil dw for aqueous LAS samples and 26 to >488 mg/kg soil dw for LAS-spiked sewage sludge samples.

In conclusion, the short-term inhibitory effects of LAS on soil microbiology were decreased in the presence of sewage sludge and by a prolonged (two to eight weeks) laboratory incubation period.

Description of key information

Microbial parameters (10 functional or structural endpoints) were reviewed but not further used by Jensen et al. (2007) in the PNECsoil assessment as originally proposed in Jensen et al. (2001). The EC10values for microbial processes observed in the laboratory ranged from <8–793 mg aqueous LAS/kg soil dry matter (DM). Microbial iron reduction was the parameter most sensitive to LAS (extrapolated value of 5 mg/kg; Jensen et al., 2001), but this endpoint was not considered relevant for aerobic agricultural soils because the extrapolation was not well justified and in conflict with experimental data conducted under more realistic conditions (field trials) later developed by Jensen et al. (2007). Furthermore, the lowest microbial effect concentrations had been observed in the case of dosing of aqueous LAS solutions, whereas in reality LAS enters soil in a sludge matrix, where bioavailability was shown to considerably mitigate toxic effects (Elsgaard et al., 2001; Gejlsbjerg et al., 2001). For example, ammonium oxidation, an important aerobic transformation process showed a lowest EC10value of 14 mg/kg for aqueous dosing, versus 68 mg/kg for LAS in a sludge matrix (Elsgaard et al., 2001). In the latter study, microbial communities also showed a strong recovery potential (Schowanek et al., 2007).

In conclusion, terrestrial toxicity to soil micro-organisms is not needed considering the higher rate of sensitivity, and thus protection afforded by, studies on plants and invertebrates exposed to LAS under more realistic test conditions (field trials).

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
35 mg/kg soil dw

Additional information

Jensen, J., Smith, S. R., Krogh, P. H., Versteeg, D. J., & Temara, A. (2007). European risk assessment of LAS in agricultural soil revisited: species sensitivity distribution and risk estimates. Chemosphere, 69(6), 880-892.