Ethyl 3,5,5-trimethylhexanoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage treatment plant Hochdahl, Germany

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

act. ingr.

Initial conc.:

5 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: no data
- Solubilising agent (type and concentration if used): Disponil NP 9.5EO 5PO
- Test temperature: 20±1°C
- pH: no data
- Continuous darkness: no data

TEST SYSTEM
- Culturing apparatus: conical shoulder bottle
- Number of culture flasks/concentration: 2 (inoculum blank: 4)
- Measuring equipment: iodometric oxygen measurement, Winkler method
- closed system


SAMPLING
- Sampling frequency: 7, 14, 21, 28 d

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: no
- Other: O2 control (mineral medium without inoculum and test substance)
- Solubilising agent control (mineral medium, solubilising agent, inoculum)

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Remarks on result:

other: 2 mg/L

Parameter:

% degradation (O2 consumption)

Value:

8

Sampling time:

28 d

Remarks on result:

other: 5 mg/L

Results with reference substance:

Sodium benzoate was degraded normally: 77% after 7 d, 84% after 14d, 88% after 21 d, 89% after 28 d

 Biodegradation:

	Concentration [mg/L]	% BOD/COD
		7 d	14 d	21 d	28 d
Test substance	2	0	1	13	4
	5	2	1	4	8
Reference substance	2	77	84	88	89

 

Oxygen consumption: 

	Concentration [mg/L]	Oxygen concentration [mg/L]
		7 d	14 d	21 d	28 d
Test substance	2	9.40 9.01	8.69 8.75	8.63 7.61	8.54 8.76
	5	8.96 8.81	8.72 8.72	8.44 8.58	8.46 8.18
Reference substance	2	6.42 6.85	5.99 6.09	6.00 5.81	6.00 5.96
Inoculum blank	---	9.16 9.18 9.27 9.25	8.89 8.79 8.84 8.88	8.83 8.89 8.83 8.86	8.99 8.98 8.96 8.90
Solubilising agent	Test conc. 1	9.03 8.80	8.77 8.79	8.82 8.82	8.72 8.88
	Test conc. 2	9.31 8.98	8.76 8.90	9.35 8.71	9.38 9.50

 

 

 

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of this test, no biodegradation of 3,5,5-Trimethyl-ethyl capronate was observed.

Executive summary:

The biodegradation of 3,5,5-Trimethyl-ethyl capronate (95% a.i.) was investigated in a study conducted according to OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) over a period of 28 days using activated sludge sampled from a municipal sewage treatment plant as inoculum. Due to the low water solubility of the test substance, Disponil NP 9.5EO 5PO was used as solubilising agent.

The biodegradation rate was determined by measurement of O2 consumption. Inoculum blank, O2 control (mineral medium without inoculum and test substance), solubilising agent control (mineral medium, solubilising agent, inoculum) and procedural/functional control with the reference substance Sodium benzoate were performed. The test was performed with 2 and 5 mg/L test substance concentration.

The functional control reached the pass level >60% after 7 d (77%).

At the end of the 28-day exposure period, the mean extent of biodegradation of the test item was 4% at 2 mg/L test item concentration and 8% at 5 mg /L test item concentration.

No significant biodegradation of 3,5,5-Trimethyl-ethyl capronate occurred under the test conditions within 28 days and the pass level for ready biodegradability of at least 60% degradation 10 day window within the 28 day period of the test was not reached.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From July 27, 2016 to August 24, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test method is according to OECD guideline 301F and it is not GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

Reference substance is not the one recommended in the guideline.

GLP compliance:

no

Remarks:

test conducted for screening purposes and therefore not GLP.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Conditions of incubation
-Concentration of test item: 100 mg/L
-Concentration of activated sludge: 30 mg/L (as concentration of suspended solid)
-Volume of test medium: 300 mL
-Incubation temperature: 25±1 °C
-Incubation duration: 28 days

Measurement for calculation of percentage biodegradation
-The degradation of the test material was assessed by the determination of biological oxygen demand (BOD) by oxygen consumption of microorganism. In addition, dissolved organic carbon (DOC) was measured at the end of the study by a total organic carbon analysis (TOC).

Parameter:

% degradation (O2 consumption)

Value:

30

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Sampling time:

28 d

Remarks on result:

other: DOC concentration in biotic and control assays after 28 days of incubation was found to be 1.1 and 1.2 mg DOC/L, respectively. The recovery rate was comparable in biotic and control assays.

Table 1 % degradation of test items

Replication number	% Biodegradation by BOD
	Day 4	Day7	Day 14	Day 21	Day 28
1	9	9	22	26	28
2	9	19	27	30	32
Mean	9	14	24	28	30

Table 2 DOC Measurement of test items and controls

Replication number	Dissolved organic carbon (DOC) at Day 28 (mg C/L)
1	1.165
2	1.124
Control 1	1.093
Control 2	1.267

Validity criteria fulfilled:

not applicable

Interpretation of results:

inherently biodegradable

Conclusions:

The test material attained 28% and 32% degradation, respectively, after 28 days. The test material can therefore considered to be not readily biodegradable under the test conditions of OECD Guideline No.301F.

Executive summary:

This study was performed to assess the ready biodegradability of the test material in a biodegradation test. The method followed was designed to be according to the OECD Guidelines for Testing of Chemicals, No.301F, July 17, 1992, “Manometric Respirometry Test”.The test material attained 28% and 32% degradation, respectively, after 28 days. The test material can therefore considered to be not readily biodegradation under the test conditions of OECD Guideline No.301F.

DOC concentration in biotic and control assays after 28 days of incubation was found to be 1.1 and 1.2 mg DOC/L, respectively. The recovery rate was comparable in biotic and control assays.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 11, 2017 to April 17, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Fresh samples of activated sludge were withdrawn on August 14th, 2017 (test run A, Mineralization and specific chemical analysis) and on November 07 th, 2017 (test run B, DOC concentration after 28 days of incubation) from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater. Since it was not necessary, the samples were not washed with mineral medium after the arrival at the laboratory but kept aerobic until use. The sludge was left for settlement for ca. one hour. Subsequently the supernatant was discarded and the concentration of suspended solids was measured in the remaining sludge. The concentration was adjusted to 4.3 g/L (test run A) and 4.8 g/L (test run B), respectively, and verified by dry mass measurement. The concentration used in the test was 29.6 mg dry mass/liter.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

including a hydrolysis product

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

A measured volume of inoculated mineral medium, containing a known concentration of test item (100 mg test item/L, respectively, giving at least 50-100 mg ThOD/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature of 22 °C ± 1°C for up to 28 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in a suitable absorbent. The amount of oxygen taken up by the microbial population during biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD.
Additionally, test item loading (total test item amount in the test vessel), dissolved test item concentration and the concentration of the dissolved hydrolysis product 3,5,5-trimethylhexanoic acid were quantified by chemical analysis in the course of the study. DOC concentration was measured at the end of the 28-day incubation period.


-Water
For the test deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions) was used. The organic carbon content was checked at regular intervals (monthly) by dissolved organic carbon (DOC) analysis. The maximum DOC content was verified in the last sampling before and the first sampling after medium preparation and was found to be 0.18 mg/L (test run A) and 0.92 mg/L (test run B), corresponding with 0.3% and 1.3% of the organic carbon content introduced by the test item (70.9 mg C per liter), respectively. According to the OECD 301 guideline, a value of 10 % should not be surpassed.

-Test item
The concentration in the test assays were 100 mg per liter mineral test medium (25 mg/250 mL, test run A; 7 mg/60 mL, test run B).

-Reference item (test run A)
Sodium benzoate was used as reference item in test run A. The concentration in the test vessels with reference item (procedural control, toxicity control) was 100 mg per liter mineral test medium (25 mg/250 mL).

-Control(s)
Blank control
The blank control consists of inoculated mineral medium only.
Abiotic control
An abiotic control without inoculum but containing test item at 100 mg per liter mineral test medium (25 mg test item/250 mL) and 10 mL/L NaN3 (10%) as sterilising agent was applied.
Toxicity control (test run A)
A toxicity control containing test item at 100 mg per liter and reference item at 100 mg per liter mineral test medium (25 mg test item/250 mL and 25 mg reference item/250 mL) was applied.

-Test vessels
Test run A
Biotic test suspension: 20 vessels containing test item (100 mg/L) and inoculum
(4 of them only for chemical analysis at test start, 16 for chemical analysis and measurement of oxygen demand)
Abiotic control: 20 vessels containing test item (100 mg/L) and a sterilising agent
(4 of them only for chemical analysis at test start, 16 for chemical analysis and measurement of oxygen demand)
Inoculum blank: 2 vessels containing only inoculum
(for measurement of oxygen demand)
Procedural control: 2 vessels containing reference item (100 mg/L) and inoculum
(for measurement of oxygen demand)
Toxicity control: 2 vessels containing test item (100 mg/L), reference item (100 mg/L) and inoculum
(for measurement of oxygen demand)

Test run B
Biotic test suspension: 2 vessels containing test item (100 mg/L) and inoculum
Abiotic control: 2 vessels containing test item (100 mg/L) and a sterilising agent
Inoculum blank: 2 vessels containing only inoculum

-Measurement devices for oxygen demand (test run A)
The incubation of the vessels took place in a water bath, adjusted on 22 °C. The measurement and recording of the oxygen demand was carried out continuously using a SAPROMAT respirometer (VOITH Inc.).

-Specific chemical analysis (test run A)
The total amount of test item available in the test vessels and the concentration of dissolved test item and the hydrolysis product 3,5,5-trimethylhexanoic acid were followed by specific chemical analysis. Sampling was done at test start, on day 2, 7, 14 and 28. On the days of measurement, four test vessels of the test assay and the abiotic control were removed from the respirometer. Two vessels were extracted by liquid/liquid extraction for quantification of the test item and hydrolysis product loading and two vessels were used for quantification of dissolved test item and hydrolysis product concentration. A GC/MS system was applied for analysis. Samples were analyzed immediately after sampling.

-DOC analysis (test run B)
DOC concentration was measured at the end of a 28 day incubation period. DOC measurement was conducted with a TC-analyzer (SHIMADZU TOC-V CPH) directly by quantification of non-purgable organic carbon (NPOC). NPOC was measured by acidifying an aqueous sample and then sparging the sample to strip off any purgeable organic and inorganic carbon. The sample was then injected into a combustion tube that contains a catalyst material. A redox reaction occured that evolved carbon dioxide gas (CO2) which was then detected by a non-dispersive infrared (NDIR) detector.

-Test procedure test run A
The test item in a concentration of about 100 mg per liter mineral medium and sodium benzoate in a concentrations of about 100 mg per liter mineral medium, were incubated with 29.6 mg dry mass inoculum per liter mineral medium in 500 mL glass vessels at a medium volume of 250 mL. The test was run for 28 days, in darkness at 22°C ± 1°C. The suspension was aerated during the whole test. Oxygen demand was carried out continuously throughout the test course.

-Test procedure test run B
The test item in a concentration of about 100 mg per liter mineral medium was incubated with 29.6 mg dry mass inoculum per liter mineral medium in 120 mL glass vessels at a medium volume of 60 mL. The test was run for 28 days, in darkness at 22°C ± 1°C. Due to the minimal oxygen demand in test run A, sealed vessels were applied in test run B and oxygen demand was not measured. Ratio of test medium and headspace is comparable to the original test run. Aim of this test run was to observe if generally any unknown dissolved organic transformation products beside the test item and the hydrolysis product 3,5,5-trimethylhexanoic acid – quantified as DOC – can be found after 28 days of incubation.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

13

St. dev.:

0.7

Sampling time:

28 d

Key result

Parameter:

% degradation (test mat. analysis)

Remarks on result:

other: Only <1 mg/L of the test item was detected from day 2. Also < 1 mg/L of the hydrolysis product was detected from day 0.

Key result

Parameter:

% degradation (DOC removal)

Sampling time:

28 d

Remarks on result:

other: DOC concentration in biotic and abiotic assays after 28 days of incubation was found to be 2.81 and 2.42 mg DOC/L, respectively (3.4 – 4.0% of nominal loading).

Details on results:

-Mineralization
The mineralization of the test substance – based on nominal loading – after 28 days of incubation in the static test was found to be 13.0% (SD = 0.7%) and 1.7% (SD = 0.7%) in the biotic test suspension and the abiotic control, respectively. The mineralization within the 10-day-window was 13.0 % in the biotic test suspension. The 10-day-window started at day 9.

The biodegradation of the item mixture in the toxicity control was found to be 42 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item can be identified as non-toxic in a ready biodegradability test. The reference item sodium benzoate was degraded to 83 % within the first 14 days.

According to the OECD guideline 301F, the test item must be considered as being not readily biodegradable under the test conditions applied.

-Primary degradation
The total recovery rate of the test substance at test start was found to be at mean 50.6 - 71.8 mg/L, corresponding with 50.6 - 71.8% of the nominal loading. After two days, total recovery rate decreased on 0.3 – 0.4% and maintained stable at 0.1 – 0.2% starting at day 7. The recovery rate was comparable in biotic and abiotic assays. With 0.3 – 0.4 mg/L, the hydrolysis product 3,5,5-trimethylhexanoic acid only was found in biotic assays until day 2. In abiotic assays and starting at day 7 also in biotic assays, concentration was < LOQ (0.206 mg/L).


-DOC concentration
DOC concentration in biotic and abiotic assays after 28 days of incubation was found to be 3.4 – 4.0% of nominal loading. The recovery rate was comparable in biotic and abiotic assays.

-Validity
The Manometric Respirometry Test fulfills the validity criteria of the guideline:
o With 1% the difference of extremes of replicate values of the removal of the test item in the biotic test assays was less than 20% at the end of the 10-day-window and at test end.
o The percentage degradation of the reference item has exceeded the pass level of 60% by day 14.
o The oxygen uptake of the inoculum blank is < 60 mg/L in 28 days and the mean pH values in all assays were inside the range of 6.0 - 8.5.

See attached a figure and tables for detailes on results

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mineralization of the test item – based on nominal loading - in the static test was found to be at mean 13.0% with a standard deviation of 0.7 % in biotic assays after 28 days.

The total recovery rate of the test item at test start was found to be at mean 50.6 - 71.8% of the nominal loading. After two days, total recovery rate decreased on 0.3 – 0.4% and maintained stable at 0.1 – 0.2% starting at day 7. The recovery rate was comparable in biotic and abiotic assays. With 0.3 – 0.4 mg/L, the hydrolysis product 3,5,5-trimethylhexanoic acid only was found in biotic assays until day 2. In abiotic assays and starting at day 7 also in biotic assays, concentration was < LOQ (0.206 mg/L). DOC concentration in biotic and abiotic assays after 28 days of incubation was found to be 3.4 – 4.0 % of nominal loading.

Executive summary:

At the Fraunhofer Institute for Molecular Biology and Applied Ecology,the biodegradation of the test item was investigated over a 28-day period in a Manometric Respirometry Test according to EC method C.4-D (440/2008/EEC)and OECD guideline 301 F (1992). The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater.

 

The test solutions were stirred in closed flasks at 22 °C ± 1°C for 28 days. The rate of degradation was monitored by measuring the quantity of oxygen required to maintain a constant gas volume in the respirometer flasks over a 28-d period. The amount of oxygen taken up by the microbial population during biodegradation of the test item at a concentration of 100 mg/L, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the ThOD (theoretical oxygen demand). In order to check the procedure, sodium benzoate was used as a degradable reference item at a concentration of 100 mg/L, along with a toxicity control at 100 mg/L the test item and 100 mg/L sodium benzoate.

 

Test item loading (total test item amount in the test vessel), dissolved test item concentration and the concentration of the dissolved hydrolysis product 3,5,5-trimethylhexanoic acid were quantified by chemical analysis. For this, aliquots of biotic and abiotic assays were analyzed at test start, day 2, 7, 14 and 28. In an additional test run under comparable test conditions, DOC concentration in biotic and abiotic assays was measured after 28 days of incubation.

 

The mineralization of the test item – based on nominal loading - in the static test was found to be at mean 13.0% with a standard deviation of 0.7 % in biotic assays after 28 days. Consequently, the mineralization within the 10-day-window was also found to be 13.0%. The 10-day-window started at day 9. The mineralization under abiotic conditions was at mean 1.7% with a standard deviation of 0.7 % after 28 days.

 

The degradation of the reference item sodium benzoate had reached 83 % within the first 14 days. The difference of extremes of replicate values of the removal of the test item at the end of the test for biotic and abiotic assays is less than 20%. Therefore, the test can be considered as valid. No inhibitory effects of the test item were observed (more than 25% degradation occurred within 14 days) in the toxicity control.

 

The total recovery rate of the test item at test start was found to be at mean 50.6 - 71.8% of the nominal loading. After two days, total recovery rate decreased on 0.3 – 0.4% and maintained stable at 0.1 – 0.2% starting at day 7. The recovery rate was comparable in biotic and abiotic assays. With 0.3 – 0.4 mg/L, the hydrolysis product 3,5,5-trimethylhexanoic acid only was found in biotic assays until day 2. In abiotic assays and starting at day 7 also in biotic assays, concentration was < LOQ (0.206 mg/L). DOC concentration in biotic and abiotic assays after 28 days of incubation was found to be 3.4 – 4.0 % of nominal loading.

Description of key information

Three studies are available to assess the biodegradation of ethyl 3,5,5-trimethylhexanoate. A supporting study conducted in 1992 showed <10% degradation, however another supporting study conducted in 2017 showed 30% degradation. Also the DOC analysis from this study showed that no significant organic carbon remained after the test period, suggesting that the disappearance of the test substance from the test system. Their reliability was assessed as reliable with restrictions because the first study lacks of detailed documentation and the second study was a non-GLP study.

 

The key study was conducted according to OECD guideline 301F under GLP condition. In addition to the O2 consumption, the chemical analysis for the test substance and a hydrolysis substance and DOC analysis were conducted to elucidate the fate of the test substance in detail. Based on the O2 consumption, the result showed 13% degradation. However, the chemical analysis showed that even from day 2, only less than 1 mg/L of the test substance was detected (nominal loading was 100 mg/L) and only less than 1 mg/L of the hydrolysis substance was detected. Moreover, DOC concentration was found to be only 4% of nominal loading. These findings suggested that either the majority of the test substance disappeared or was converted to insoluble unknown organic substances. Thus, an additional chemical analysis and a study were conducted (detailed in the "additional information" in this endpoint summary).

 

First, the additional SCAN chemical analysis was conducted. It demonstrated that no other substances than the test substance were detected at day 2, suggesting that test substance was not converted to any measurable metabolites. Second, the additional study and the analysis with the sealed vials were conducted. It showed that the test substance was detected in the headspace of both biotic and abiotic samples, suggesting that the test substance volatized.

 

According to the OECD Guidance Document No,23 (2000), if H (the Henry's law constant) is greater than 100 Pa.m3/mol, more than 50% of the substance could be lost from the water phase in 3 -4 hours. Based on the experimental data of water solubility, vapour pressure and molecular weight, the Henry's law constant of the test substance was calculated as 440 Pa.m3/mol, which backs up our findings.

 

On the other hand, the degradation of the study showed 13% degradation. According to the chemical analysis on day 0, 12% of the test substance was dissolved in the test solution, which is equivalent to the % degradation result. Also both the structurally similar substance, 3,5,5-trimethylhexyl acetate (CAS: 58430-94-7) and the hydrolysis substance of the test substance, 3,5,5-trimethylhexanoic acid (CAS:3302-10-1) are readily biodegradable according to the registered dossiers in REACH. Therefore, it can be assumed that, the result didn't come from the degradation of the partial structure, but the dissolved fraction of the test substance was fully mineralized by the microorganisms within 2 days.

 

Based on all the findings and related information, it could be concluded that the dissolved test substance was biodegraded and the rest of the test substance disappeared by volatilization, which leaded to the only13% of biodegradation. Thus, it could be considered that the registered substance possessed the potential to be biodegraded inherently.

 

It should be noted that a proper test method will be needed to evaluate the true biodegradation potential of this registered substance. 

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Short Report : Additional tests (non-GLP) to the GLP study

<Scanning for metabolites>

 

Furthermore an inoculum total volume extraction sample from day two in main study (GLP) was run in SCAN mode to check for any measurable metabolite or hydrolysis products other than the test substance and 3,5,5 trimethyl hexanoic acid. In the obtained chromatogram, no substance other than the test substance could be determined.

 

<Headspace analysis>

 

Additionally to the main manometric respirometry test, other measurements were run to test the headspace above the sample for the test substance. These tests indicate that the substance is highly volatile and readily evaporates.

 

Method:

In serum flasks, 50 µL of the test substance was spiked into 50 mL biotic test suspension samples and abiotic control samples respectively. The flasks were sealed with crimped caps. After 28 days of incubation, a 25µL sample from the headspace was taken with a syringe and manually injected into the Gas Chromatograph. Measurements were carried out in SCAN mode to check for the test substance and other substances that might have evaporated into the headspace.

Additionally Samples were prepared in extra serum flasks to check the amount of substance in the headspace after 2 and 6 days of incubation.

 

Results:

After 28 days of incubation, the biotic test suspension samples showed no substance in the headspace, whereas the abiotic control had so much substance in the headspace that the GC system was completely overloaded.

The additional samples showed that the biotic control had lower amounts of the test substance in the headspace than the abiotic samples after two days. The substance significantly increased in the headspace of the biotic samples over the amount of 4 days, whereas it was fairly constant in the abiotic control samples.