3,7-dimethylocta-1,3,6-triene

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information is used for read across to Ocimene.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands (29-12-2008). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week with moist air.

Duration of test (contact time):

28 d

Initial conc.:

29.6 mg/L

Based on:

test mat.

Initial conc.:

26 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Nutrient solution and stock solutions: 85 mg KH2PO4, 217.5 mg K2HPO4, 334 mg Na2HPO4·2H2O, 5 mg NH4Cl, 22.5 mg MgSO4·7H20, 27.5 mg CaCl2, 0.25 mg FeCI3·6H20. 1-Octanol was added to the vessels using a stock solution in dichloromethane of 1.0 g/ L. Myrcene is a poorly soluble substance in water. The test substance was directly added to the vessels.
- Suspended solids concentration: 4 mg DW/L

TEST SYSTEM
- Test vessels: the test was performed in 120 mL serum flaks with butyl septa and crimp-on aluminum seals (control and reference) or Mininert valves (test substance). The volume of the liquid phase was 80 mL.

TEST PROCEDURE
The carbon dioxide headspace test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (2005). Use was made of 30 vessels containing only inoculum, 30 vessels containing test substance and inoculum, and 9 vessels containing 1-octanol and inoculum. One additional bottle of each series was included to allow determination of the pH at day 28. 1-Octanol in dichloromethane (2.5 mL) was directly added to bottles. The solvent was subsequently allowed to evaporate in a ventilated hood for 24 hours. Volumes of 3 µL of the test substance were added to the vessels. The amounts of test substance added were checked by weighing 10 times, 3 µL of test substance.
The concentrations of the test substance and 1-octanol in the vessels were 29.6 and 31.3 mg/L, respectively, representing organic carbon concentrations of 26.0 and 23.1 mg/L respectively. The inoculum was diluted to 4 mg DW/L in the test vessels. 80 mL of each of the prepared solutions was dispensed into the respective group of test vessels. The zero time vessels were analyzed for total inorganic carbon using the TOC apparatus upon adding 1 mL of a freshly prepared 7 M NaOH solution with 1 mL plastic syringes (see 'Details on analytical methods'). The remaining vessels were incubated in the dark. The biological reactions in the test vessels were stopped by injecting 1 mL of a 7 M NaOH solution through the septum or Mininert valves. Before analysis of the inorganic carbon, the test vessels were shaken for at least one hour. Next, the particles in the vessels were allowed to settle and a suitable sample was withdrawn from the vessel with a syringe for analysis. The conversion of carbon dioxide to carbonate with sodium hydroxide should result in a negligible carbon dioxide concentration in the headspace. This was checked by adding 10 and 20 mg/L of carbonate-carbon to the minera) salts medium in the test vessels (in triplicate). A control without addition of carbonate was included. These test vessels were incubated at 20°C on a rotary shaker at 180 rpm for 24 hours. Next, sodium hydroxide (1 mL, 7 M) was added and the vessels were shaken for one hour. The inorganic carbon was measured in the alkaline medium using the TOC analyzer.

SAMPLING
Triplicate vessels of all series were withdrawn for analyses of the carbon dioxide formed at day 3, 7, 10, 14, 17, 21, and 28.

ADDITIONAL MEASUREMENTS
- pH and temperature: The pH was measured using a Knick 765 calimatic pH meter. The temperature was measured with a Keithley Digital thermometer.

Reference substance:

other: 1-octanol

Remarks:

31.3 mg/L (based on test mat.) / 23.1 mg/L (based on TOC)

Test performance:

VALIDITY
The mean carbonate-carbon concentrations and standard deviations in vessels containing the nutrient solution, nutrient solution and 10 mg/L carbonate-carbon and nutrient solution and 20 mg/L of inorganic carbon were 2.1 ± 0.7, 14.0 ± 0.3 and 24.1 ± 1.1 mg/L, respectively. The carbon added was therefore recovered for 119% and 110% at 10 and 20 mg/L carbonate-carbon, respectively. This demonstrates that carbon dioxide was removed from the headspace adequately. The validity of the test is demonstrated by an increase of the inorganic carbon content in the blank controls of 3.1 mg/L at day 28 which is ≤ 15% of the inorganic carbon added initially as test substance. The viability of the inoculum and validity of the test were supported by the results of the reference substance.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

73

Sampling time:

28 d

Details on results:

Myrcene is biodegraded 73% at day 28 in the carbon dioxide headspace test (see 'Any other information on results incl. tables'). Myrcene only fulfilled one criterion for ready biodegradable compounds i.e. a biodegradation percentage in excess of 60 within 28 days. However, the pass level of 60% was not reached within 10 days upon achieving 10% biodegradation. Failing the 10-day window criterion may be caused by 1) the toxicity of myrcene to microorganisms and/or 2) the use of test vessels which were sacrificed and/or 3) the poor water solubility of myrcene. The possible toxicity of terpenes has been reviewed by Sikkema et al (1995). The "linear" biodegradation curve with biodegradation percentages not always increasing with time may be explained with the poor water solubility of myrcene and the set-up of the test (sacrificing test bottles), respectively. The 10-day time window criterion was developed on the assumption that compounds are degraded according to "standard" logarithmic growth curves found with water soluble compounds. The time-window should therefore be ignored as a pass fait criterion for myrcene. Myrcene is classified as readily biodegradable only based on the biodegradation percentage of 73% at day 28.

Results with reference substance:

The biodegradation of the reference compound was 89% on Day 14.

Table: Inorganic carbon concentration (mg/L) in the aqueous phase (alkaline) of the test vessles

Time (days)	Inorganic carbon concentration (mg/L) [mean values, N=3]
	CLc	CLt	CLa
0	1.6	2.0	2.0
3	2.3	5.3
7	3.0	10.1	22.1
10	2.0	8.0
14	2.2	5.9	22.6
17	2.8	14.4
21	2.4	13.5
28	4.7	23.8

CLc: Mineral nutrient solution without test material but with inoculum.

CLt: Mineral nutrient solution with test material (29.6 mg/L), and with inoculum

CLa: Mineral nutrient solution with 1-octanol (31.3 mg/L) and with inoculum.

Table: Total inorganic carbon (TIC) formation (mg/L) and the percentages for biodegradation

of myrcene (TIC/ThIC) and 1-octanol (TIC/ThIC) in the carbon dioxide headspace test.

Time (days)	(mg/L)		Biodegradation (%)
	Test	1-Octanol	Test	1-Octanol
0	0.4	0.4
3	3.0		12
7	7.1	19.1	27	83
10	6.0		23
14	3.7	20.4	14	89
17	11.6		45
21	11.1		43
28	19.0		73

Validity criteria fulfilled:

yes

Remarks:

see 'Test performance'.

Interpretation of results:

readily biodegradable

Conclusions:

The substance showed >60% biodegradation within the 28-day test period of an OECD TG 310 study, although the 10-day window criterion was not fulfilled.

Executive summary:

The biodegradation potential of the substance in water was determined in a screening study according to OECD TG 310 (Headspace Test) and in compliance with GLP criteria. In this study 29.6 mg/L test substance was inoculated with activated sludge from a domestic wastewater for 28 days under aerobic conditions. During the incubation period the CO2 evolution was measured and biodegradation expressed as percentage of the theoretical CO2 production. Results for this experiment showed that there was 73% mean biodegradation in the bottles containing test substance on day 28. On day 14, biodegradation of the reference compound 1-octanol was 89%. The 10-day window criterion was not fulfilled (12% biodegradation on day 3 and 14% on day 14). The "linear" biodegradation curve with biodegradation percentages not always increasing with time may be explained with the poor water solubility of myrcene and the set-up of the test (sacrificing test bottles), respectively. The 10-day time window criterion was developed on the assumption that compounds are degraded according to "standard" logarithmic growth curves found with water soluble compounds. The time-window should therefore be ignored as a pass fail criterion for myrcene. Myrcene is classified as readily biodegradable only based on the biodegradation percentage of 73% at day 28.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

Read across justification is presented in the Endpoint summary. The accompanying file is also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

% degradation (CO2 evolution)

Value:

73

Sampling time:

28 d

Interpretation of results:

readily biodegradable

Description of key information

Ocimene: Readily biodegradable (Read across from Myrcene OECD TG 310)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

No data is available for the substance itself. Therefore read-across is performed to the close structural analogue Myrcene (CAS# 123-35-3). First the experimental information on Myrcene is presented and thereafter the read across justification.

Myrcene and its ready biodegradability

The biodegradation potential of Myrcene in water was determined in a screening study according to OECD TG 310 (Headspace Test) and in compliance with GLP criteria. In this study 29.6 mg/L test substance was inoculated with activated sludge from a domestic wastewater for 28 days under aerobic conditions. During the incubation period the CO2 evolution was measured and biodegradation expressed as percentage of the theoretical CO2 production. Results for this experiment showed that there was 73% mean biodegradation in the bottles containing test substance on day 28. On day 14, biodegradation of the reference compound 1-octanol was 89%. The 10-day window criterion was not fulfilled (12 % biodegradation on day 3 and 14% on day 14). The "linear" biodegradation curve with biodegradation percentages not always increasing with time may be explained with the poor water solubility of Myrcene and the set-up of the test (sacrificing test bottles), respectively. The 10-day time window criterion was developed on the assumption that compounds are degraded according to "standard" logarithmic growth curves found with water soluble compounds. The time-window should therefore be ignored as a pass fail criterion for Myrcene. Myrcene is classified as readily biodegradable only based on the biodegradation percentage of 73% at day 28. Myrcene is readily biodegradable and therefore Ocimene will be assessed as readily biodegradable as well.

Biodegradation of Ocimene (CAS #13877-91-3) based on read-across from data available for Myrcene (CAS #123-35-3).

Introduction and hypothesis for the analogue approach

Ocimene has a poly-unsaturated alkene chain backbone consisting of 8 carbon atoms containing 3 double bonds and methyl substituents on the 3^rdand 7^thposition.For this substance there are no experimental biodegradation data available. Biodegradation information may be generated by other means, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the ready biodegradability of Ocimene the analogue approach is selected because for a closely related analogue, Myrcene, ready biodegradation information is available which can be used for read-across.

Hypothesis: Ocimene has the same biodegradation potential as Myrcene. The structural difference between these molecules is the position of a double bond.These minor structural differences are not expected to significantly affect the biodegradability.

Available information: For Myrcene a ready biodegradation study was performed according to OECD TG 310 and in compliance with GLP criteria (IFF, 2009). In this study Myrcene was biodegraded by 73% at day 28 in the Headspace Test.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the data matrix, including physico-chemical properties and available ecotoxicological information.

Purity / Impurities

Ocimene is a multi-constituent consisting of isomers E (66%, CAS 3779-61-1) and Z (29%, CAS 3338-55-4) and one impurity1-methylidene-4-(prop-1-en-2-yl)cyclohexane(1.5%, CAS499-97-8).Analogue approach justification

According to Annex XI 1.5 read-across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. The ECHA guidance (2017, RAAF) has been considered for this read across.

Structural similarities and differences: Ocimene and Myrcene have the same alkene backbone, with 3 double bonds which can be conjugated and are branched with a methyl group on two positions. The one difference is that Ocimene (trans) has the double bond in the long alkene chain, while Myrcene has the double bond attached to the methyl group branching from the alkene chain. For both substances this results in a conjugated bond with the primary double bond (at the top of the structure, see data matrix).

Bioavailability:In view of the similarity in structure, both being liquids, having the same molecular weight and similar physico-chemical properties the availability for the micro-organisms will be the same. The measured log Kow of Ocimene is slightly higher compared to Myrcene but this is thought to be due to experimental variability as the predicted values are 4.8 for Ocimene and 4.9 for Myrcene.

Biodegradation profile:Poly-unsaturated alkene chains are expected to be readily biodegradable in view of their similarity with e.g. unsaturated fatty acids. This is further supported with BIOWIN predictions.

BIOWIN predictions: BIOWIN (v4.10) predictions are made for both Ocimene and Myrcene to provide further insight on the biodegradation potential of these substances and the possible effect of structural differences on biodegradation. The BIOWIN modules 5 (‘MITI-linear’) and 6 (‘MITI-non-linear’) are used because these two models are based on ready biodegradability tests, which is the endpoint to be predicted.

Table 1 BIOWIN predictions for Ocimene and Myrcene and the contribution of structural fragments

Substane	Ocimene			Myrcene
Fragment	No. of occurrences	BIOWIN 5	BIOWIN 6	No. of occurrences	BIOWIN 5	BIOWIN 6
Methyl [-CH3]	3	0.0012	0.0583	2	0.0008	0.0389
-CH2- [linear]	1	0.0494	0.4295	2	0.0988	0.8590
-C=CH [alkenyl hydrogen]	5	0.0309	0.1426	6	0.0371	0.1711
MW parameter	-	-0.4053	-3.9330	-	-0.4053	-3.9330
Equation constant	-	0.7121	-	-	0.7121	-
RESULT	-	0.3884	0.3150	-	0.4436	0.4162

 

It can be seen that the different fragment scores for Ocimene and Myrcene are in the same order of magnitude and that the minor structural differences between the two substances are not likely to significantly influence the biodegradation potential.

Uncertainty of the prediction: Myrcene is predicted to have slightly higher potential for biodegradation which is based mainly on the linear ‘-CH2-’ as compared to the additional methyl group in Ocimene. As however in the biodegradation screening study with Myrcene the ready biodegradation cut-off was well exceeded (73% biodegradation compared to 60% cut-off according to OECD TG 310) a similar outcome can be expected for Ocimene from such a screening test. Therefore the data derived for Ocimene can be considered sufficiently reliable.

Data matrix: The data matrix is presented at the end of this section.

Conclusions for environmental fate and classification and labelling

Myrcene is readily biodegradable and therefore Ocimene will be assessed as readily biodegradable as well.

Data matrix presenting the information to read-across for Ocimene from Myrcene

Common name	Ocimene	Myrcene
Chemical name	(3E)-3,7-dimethylocta-1,3,6-triene, (3Z)-3,7-dimethylocta-1,3,6-triene	1,6-Octadiene, 7-methyl-3-methylene-
Chemical structures	3E                             3Z
Cas no	3779-61-1 and 3338-55-4
CAS no generic	13877-91-3	123-35-3
REACH registered	2018	Registered
EINECS	237-641-2	204-622-5
Empirical formula	C10H16	C10H16
Molecular weight	136.24	136.24
Physical state	Liquid	Liquid
Melting point	<-20 °C (IFF measured)	<-80 °C (ECH dissemination site)
Boiling point	184.1 °C (IFF measured)	165oC (ECHA dissemination site)
Vapour pressure	220.7 Pa (at 24 °C) (IFF measured)	267 Pa (ECHA dissemination site)
Water solubility	14.5 (at 24 °C) (IFF measured)	5.1 (ECHA dissemination site)
Log Kow	5.4 (at 25 °C) (IFF measured) 4.8 (EpiSuite prediction)	4.8 (ECHA dissemination site) 4.9 (EpiSuite prediction)
Biodegradation	Read across	Readily biodegradable (OECD TG 310)