Tricyclodecanedimethanol

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Transformation products:

not measured

Reference 2

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental phase: 2021-07-13 to 2021-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

Adopted April 13th 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Remarks:

Different strategies of synthesis were considered for 14C-labelling, and 4 specialized laboratories inquired: no laboratory was able to perform the task due to the necessary high pressure.

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

Natural surface water (field fresh sampled) of the river ‘LEINE’, containing a natural concentration of suspended matter of approx. 13 mg DW/L.

Details on source and properties of surface water:

See section "Any other information on materials and methods incl. tables".

Details on source and properties of sediment:

not applicable

Details on inoculum:

The water was collected from the surface of the river Leine (approx. upper 20 cm water) at an undisturbed recess. Furthermore, temperature, pH value and O2 concentration of the water were measured at field sampling (see details on parameters in section "Any other information on materials and methods incl. tables").
The water was brownish and turbid. Floating matter was removed by filtration (1 mm mesh). Suspended solids were reduced by sedimentation overnight and decantation of the water. The suspended solids concentration was adjusted by adding 200 mL of the concentrated suspended solids to approx. 30 L decanted surface water.

Acclimation:
Until test start, the prepared surface water was acclimated for six days under test conditions at 12 ± 2 °C in the dark with aeration.

Duration of test (contact time):

65 d

Initial conc.:

100 µg/L

Based on:

test mat.

Remarks:

Because the multi-constituent test item is constituted by ca. 1/3, each of three main structural isomers, the concentration per isomer is ca. 33.3 µg/L.

Initial conc.:

20 µg/L

Based on:

test mat.

Remarks:

Because the multi-constituent test item is constituted by ca. 1/3, each of three main structural isomers, the concentration per isomer is ca. 6.7 µg/L. This agrees with the guideline recommendation of less than 10 µg/L for the lower concentration.

Parameter followed for biodegradation estimation:

test mat. analysis

other:

Details on study design:

1) Type and Purpose of the Study:
Laboratory shake flask batch test to determine rates of aerobic biodegradation (primary transformation and formation of major transformation products) in aerobic natural surface water over a period of 65 days.

2) Test System and Pretreatment:
- Test system: Natural surface water (field fresh sampled) of the river ‘LEINE’, containing a natural concentration of suspended matter of approx. 13 mg DW/L. The sampling site was selected with respect to the regional biological and chemical water quality maps (interactive online version) of the Lower Saxony Water Management, Coastal Defence and Nature Conservation Agency (German: NLWKN) to comply with requirement of OECD TG 309. The surface water meets the recommendations of the guideline.
- Collection / Sampling: The water was collected from the surface of the river (approx. upper 20 cm water) at an undisturbed recess. Furthermore, temperature, pH value and O2 concentration of the water were measured at field sampling.
 
3) Experimental Procedure
3.1) Reference control
Reference item Aniline sulfate, [14C(U)]-,
Radiochemical purity 99.2%
Specific activity 110 mCi/mmol
Replicates Duplicates
Test concentration 10 µg/L

3.2) Test Groups
3.2.1) Test Item TCD Alcohol DM
- Nominal test item concentration: 20 µg/L and 100 µg/L
The lowest test item concentration of 20 µg/L test material was selected to assure an accurate kinetic analysis for the degradation kinetics of all three isomers present in the test item. Because the multi-constituent test item is constituted by ca. 1/3, each, of three main structural isomers, the concentration per isomer is ca. 6.7 µg/L. This agrees with the guideline recommendation of less than 10 µg/L for the lower concentration.
- Stock solution 10 g/L in methanol
- Application solution 100 mg/L, stock solution diluted 1:100 in ultrapure water.
- Solvent Methanol / Ultrapure Water; the amount of methanol was minimized as far as possible (0.0002% at 20 µg/L; 0.001% at 100 µg/L, % vol/vol)
- Pretreatment None

3.2.2) Blank control Surface water (test system) and 1.7 µL methanol/170 mL test solution, without test and/or reference item.

3.2.3) Sterile control Sterilized surface water with TCD Alcohol DM at test concentration. For sterilization, the water was autoclaved once (121 °C, 20 min.) prior to application.

3.2.4) Sterile Blank Control Sterilized surface water (test system) and 1.7 µL methanol/170 mL test solution, without test and/or reference item.
3.2.5) Solvent control Test water and reference item, treated with the same amount of methanol as used for the application of the test item.

4) Test Procedure
Duration 65 days
Application At application, appropriate volumes of the test item application solution were pipetted directly into the test medium of each replicate. For exact volumes see See section "Any other information on materials and methods incl. tables".
Test vessels 250 mL gastight bottles, closed
Test medium Natural surface water of the river ‘Leine’.
Volume of the test medium 170 mL
Replicates:
- Test item replicates: Duplicates per sampling and per concentration, in total 28 replicates were prepared for each concentration.
- Sterile controls: Duplicates per sampling and per concentration, in total 16 replicates were prepared for each concentration.
- Blank controls: Single, in total 20 replicates were prepared.
- Sterile blank controls: Single, in total duplicates were prepared.
- Reference control and solvent control: Duplicates, in total 6 replicates were prepared for each treatment.
Incubation
- Nominal: 12 ± 2 °C in the dark
- Actual: 10.5 – 14.0 °C in the dark
Agitation Continuous stirring at approx. 120 rpm to maintain particles and microorganisms in suspension and to facilitate oxygen transfer from the headspace to the liquid.

5) Type and Frequency of Measurements
Temperature The incubation temperature was recorded continuously throughout the test.
pH and Oxygen Measurements of pH and oxygen concentration in the test system (Blank control) were done on day 0, 3, 9, 21, 30, 44, 51, 58 and 65.
Transformation:
- Sampling for determination of the transformation was carried out directly after application and at 10 additional sampling points. The sampling points were chosen to enable the establishment of the pattern of decline of the test item. Sampling was done after application (day 0) and at day 1, 2, 3, 10, 20, 30, 44, 51, 58 and day 65.
- Two replicates per test item concentration were harvested at each sampling time for analysis of test item and metabolites.
- Sterile controls were analysed in parallel at day 0 and day 3. Blank controls and sterile blank controls were analysed at test start to verify the absence of the test item in the used surface water and as background correction for the metabolite analysis.
- The amount of test item and degradation products in the test solution and sterile test item solution was determined by a targeted analytical method for the test item and an untargeted method for metabolites by LC-HRMS methods. Additionally, the headspace of the test item replicates was sampled by purge and trap on polyurethane (PU) foams. Formation of volatile transformation products was not indicated: therefore, elution of the foams and analysis was not necessary.
- For the reference control and solvent control, sampling for mineralization was stopped once degradation was finished. Residual activity in the test solution and dissolved 14-CO2 (after acidification of test solution) were determined by LSC.

6) Evaluation
- The amounts of test item and the transformation product TCD Diacid were given as % of nominal (applied) initial test item concentration for each sampling time. Details about the evaluation of the transformation product TCD Monoacid are given in section "Any other information on materials and methods incl. tables" (analytical method).
- The DT50 and DT90 trigger endpoint determination was calculated based on the recommendations of the Guidance Document on Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on Pesticides in EU Registration (FOCUS).
- Software Calculations were carried out using the following software:
Excel, MICROSOFT CORPORATION; CAKE (COMPUTER ASSISTED KINETIC EVALUATION, Version 3.3), Tessella Ltd.

Reference substance:

aniline

Remarks:

Aniline sulfate, [14C(U)]- Batch number 373-002-110-A-20141024-PVA Radiochemical purity 99.2% Specific activity 110 mCi/mmol Test concentration 10 µg/L

Compartment:

natural water: freshwater

% Recovery:

102.5

St. dev.:

7.5

Remarks on result:

other: Mean recovery days 0, 20, 30, 44, 58, and 65. At days 1, 2, 3, and 10 recovery was lower because TCD Monoacid was present in relevant amounts (no absolute quantification possible, only increase/decrease).

Parent/product:

parent

Compartment:

water

Remarks:

Parental concentration 100 µg/L

Key result

% Degr.:

99.3

St. dev.:

0.6

Parameter:

test mat. analysis

Sampling date:

2021

Sampling time:

2 d

Remarks on result:

other: At day 2, concentrations for TCDM1 and TCDM2 were below the LOQ

Parent/product:

product

Compartment:

water

Remarks:

Parental concentration 100 µg/L; peak concentration of TCD Monoacid observed at day 2

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

One replicate on day 10 was below the limit of detection.

Sampling date:

2021

Sampling time:

10 d

Remarks on result:

other: i.e. 90% degradation within 8 days (day 2 to day 10)

Parent/product:

parent

Compartment:

water

Remarks:

Parental concentration 20 µg/L

Key result

% Degr.:

97.3

St. dev.:

2

Parameter:

test mat. analysis

Remarks:

Isomer-specific results: TCDM-1: 97%; TCDM-2: 99.7%; TCDM-3: 95.3%

Sampling date:

2021

Sampling time:

1 d

Remarks on result:

other: At day 2, all measured concentrations were below the limit of quantification for all three structural isomers.

Parent/product:

product

Compartment:

water

Remarks:

Parental concentration 20 µg/L; peak concentration of TCD Monoacid observed on day 2

% Degr.:

57

Parameter:

test mat. analysis

Sampling date:

2021

Sampling time:

3 d

Remarks on result:

other: i.e. 57% degradation within 24 h (day 2 to day 3)

Parent/product:

product

Compartment:

water

Remarks:

Parent initial concentration 20 µg/L; peak concentration of TCD Monoacid observed on day 2

% Degr.:

ca. 100

Parameter:

test mat. analysis

Remarks:

All concentrations below LOD

Sampling date:

2021

Sampling time:

10 d

Remarks on result:

other: i.e. 100% degradation within 8 days (day 2 to day 10)

Key result

Compartment:

natural water: freshwater

DT50:

>= 0.121 - <= 0.235 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Parental concentration 20 µg/L: the range is due to specific results for single isomers (TCDM 1-3)

Key result

Compartment:

natural water: freshwater

DT50:

>= 0.585 - <= 0.669 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Parental concentration 100 µg/L; the range is due to results specific for single isomers (TCDM 1-3)

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

Details on transformation products:

Transformation product #1 - TCD-Monoacid
- Formation: Product of the first biological oxidation step of TCD Alcohol DM, being an isomeric mixture, corresponding to parental isomers.
- Maximum occurrence: at both parental concentrations (20 and 100 µg/L), peak concentration was found at day 2 of the study.
- Decline during test: decline was rapidly, with 100% and 90% degraded at day 10 (i.e. after 8 days) for parental concentrations of 20 and 100 µg/L, respectively.
- Maximum concentration: absolute quantification of TCD-Monoacid was impossible due to the lack of an authentic standard. Based on the mass balance being nearly 100% for days when TCD-Monoacid was not detectable (days 0, 20-65), concentration at day 2 amounted to ca. 90% of parent both, for 100 µg/L and 20 µg/L initial concentration of TCD Alcohol DM.


Transformation products #2, #3, #4 - TCD Diacid 1-3
- Formation: Product of the second biological oxidation step of TCD Alcohol DM, structural isomers corresponding to parental isomers of TCD Alcohol DM.
- Maximum occurrence: at 20 as well as 100 µg/L initial test material concentration, after 20 days 100% of parent TCD Alcohol DM was transformed to TCD Diacid, independently of the specific parental isomers.
- Decline during test: Between day 20 and day 65 there was only a slight but not significant decline of TCD Diacid 1-3.
- Maximum concentration: highest concentrations were determined at day 20 for both, 100 µg/L and 20 µg/L initial concentration of TCD Alcohol DM. 112.8% and 112% (of applied TCD Alcohol DM) were determined for 20 and 100 µg/L initial concentration.


- Plots on decline and formation of parent and transformation products: see attached illustration!
- Pathway for transformation: see attached illustration!

Evaporation of parent compound:

no

Remarks:

Firstly, TCD Alcohol DM is not volatile. Secondly, the test was performed in closed flasks with sampling of the headspace of the test item replicates by purge and trap on polyurethane (PU) foams, such that volatilization losses can be excluded.

Volatile metabolites:

no

Remarks:

Mass balance was approximately 100%, such that any occurrence of volatile metabolites can safely be excluded.

Residues:

no

Remarks:

No residues of TCD Alcohol DM could be determined any more after 20 days and beyond, irrespective of the initial concentration.

Details on results:

Measurements of pH and oxygen concentration in the test system (blank control) were done at day 0, 3, 9, 21, 30, 44, 51, 58 and day 65. The oxygen concentration was in the range 9.01 – 10.14 mg O2/L and the pH was in the range 6.22 – 8.55.

For further details, see section "Any other information on results incl. tables" as well as attached files.

Results with reference substance:

Mineralisation of the reference item Aniline sulphate, [14C(U)]- was already > 50% after 6 days and >= 65% after 27 days both, in the reference control and the solvent control. The high mineralization indicates that the surface water contained an active microbial population.

Transformation of TCD Alcohol DM:

20 µg/L TCD Alcohol DM

		% of Applied TCD Alcohol DM (molar^#)
		Day
	Repl.	0	1	2	3	10	20	30	44	58	65
TCDM1	1	94.4	3.0	1.0	0.3	0.3
	2	96.5	3.0	0.3	0.3	0.3
	Mean	95.4	3.0	0.6	0.3	0.3
TCDM2	1	91.2	0.3	0.3	0.3	0.3
	2	93.3	0.3	0.3	0.3	0.3
	Mean	92.3	0.3	0.3	0.3	0.3
TCDM3	1	93.5	4.8	1.4	0.1	0.1
	2	93.3	4.7	1.6	0.1	0.1
	Mean	93.4	4.7	1.5	0.1	0.1
Mean TCDM	1	93.0	2.7	0.9	0.2	0.2
	2	94.4	2.7	0.7	0.2	0.2
	Mean	93.7	2.7	0.8	0.2	0.2
Mean TCD Diacid^*	1		7.0	13.8	14.3	52.3	116.6	109.6	103.1	95.4	100.1
	2		4.8	8.7	20.1	62.7	109.0	107.6	106.6	96.0	98.6
	Mean		5.9	11.3	17.2	57.7	112.8	108.6	104.9	95.7	99.3
Mass Balance	1	93.0	9.7	14.7	14.5	52.5	116.6	109.6	103.1	95.4	100.1
	2	94.4	7.5	9.4	20.3	62.9	109.0	107.6	106.6	96.0	98.6
	Mean	93.7	8.6	12.1	17.4	57.7	112.8	108.6	104.9	95.7	99.3
		TCD Monoacid Area Quotient
		Day
	Repl.	0	1	2	3	10	20	30	44	58	65
TCD Mono-acid 1	1	n.d.	0.195	0.772	0.183	n.d.	n.d.
	2	n.d.	0.181	0.166	0.208	n.d.	n.d.
	Mean	-	0.188	0.469	0.196	-	-
TCD Mono- acid 2	1	n.d.	0.135	0.639	0.179	n.d.	n.d.
	2	n.d.	n.d.	0.188	0.193	n.d.	n.d.
	Mean	-	0.067	0.413	0.186	-	-

Repl. = Replicate;  * = mean of 3 isomers; # = molar mass ratio taken into account for calculation of %; n.d. = not detected; Italic values = measured values < LOQ/LOD

 

100 µg/L TCD Alcohol DM:

		% of Applied TCD Alcohol DM (molar^#)
		Day
	Repl.	0	1	2	3	10	20	30	44	51	58	65
TCDM1	1	103.4	43.2	0.5	0.5	0.1
	2	96.7	45.6	0.5	0.2	0.1
	Mean	100.0	44.4	0.5	0.4	0.1
TCDM2	1	101.9	34.5	0.1	0.1	0.1
	2	93.3	36.9	0.1	0.1	0.1
	Mean	97.6	35.7	0.1	0.1	0.1
TCDM3	1	96.0	42.0	1.3	1.1	0.03
	2	96.6	45.7	1.5	0.6	0.03
	Mean	96.3	43.9	1.4	0.8	0.03
Mean TCDM	1	100.4	39.9	0.6	0.5	0.04
	2	95.5	42.7	0.7	0.3	0.04
	Mean	98.0	41.3	0.7	0.4	0.04
Mean TCD Diacid^*	1		3.8	8.1	10.3	44.5	112.5	110.1	105.7	99.9	105.7	107.3
	2		2.4	8.3	11.1	40.9	111.5	111.3	105.4	106.6	108.1	110.1
	Mean		3.1	8.2	10.7	42.7	112.0	110.7	105.6	103.2	106.9	108.7
Mass Balance	1	100.4	43.6	8.7	10.8	44.5	112.5	110.1	105.7	99.9	105.7	107.3
	2	95.5	45.2	9.0	11.4	40.9	111.5	111.3	105.4	106.6	108.1	110.1
	Mean	98.0	44.4	8.9	11.1	42.7	112.0	110.7	105.6	103.2	106.9	108.7
		TCD Monoacid Area Quotient
		Day
	Repl.	0	1	2	3	10	20	30	44	51	58	65
TCD Mono-acid 1	1	0.159	0.771	0.972	0.722	0.109	n.d.
	2	0.246	0.697	0.987	0.929	n.d.	n.d.
	Mean	0.202	0.734	0.980	0.825	0.055	-
TCD Mono- acid 2	1	n.d.	0.624	0.825	0.678	0.161	n.d.
	2	0.230	0.623	0.953	0.896	0.101	n.d.
	Mean	0.115	0.623	0.889	0.787	0.131	-

Repl. = Replicate;  * = mean of 3 isomers; # = molar mass ratio taken into account for calculation of %; n.d. = not detected; Italic values = measured values < LOQ/LOD

 

Sterile controls:

		% Applied TCD Alcohol DM
		20 µg/L		100 µg/L
		Day
	Repl.	0	3	0	3
TCDM1	1	98.5	102.7	107.8	101.2
	2	107.4	95.0	103.3	104.7
	Mean	102.9	98.8	105.6	102.9
TCDM2	1	101.8	105.1	105.7	105.7
	2	90.6	99.0	94.1	106.3
	Mean	96.2	102.1	99.9	106.0
TCDM3	1	98.9	104.5	100.5	104.4
	2	101.6	106.4	96.1	108.8
	Mean	100.3	105.5	98.3	106.6
Mean TCDM	1	99.7	104.1	104.7	103.8
	2	99.9	100.2	97.8	106.6
	Mean	99.8	102.1	101.2	105.2

Repl. = Replicate

 

Kinetic evaluation

The kinetic evaluations were done based on the FOCUS guidance document on estimating persistence and degradation kinetics.

The kinetic models were chosen based on the following criteria:

• Visual assessment of the fitted and observed data versus time


• Visual assessment of the residuals


• Estimation of the error percentage at which the chi-squared-test was passed

DT_x values were calculated with single first order kinetics (SFO). Reports of the kinetic fit are given in the attached documentation.

DT_x values for the metabolite TCD Monoacid were derived conservatively by fitting the decline of the metabolite concentration from the maximum onwards. The highest occurrence of TCD Monoacid was fixed at 100 % correspondingly; the respective day was set to day 0.

Data of Kinetic Fit and DTx Values of TCD Alcohol DM and TCD Monoacid

20 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model	Single First Order (SFO)
Chi-square	1.03	nd	2.06	0.13
DTX values in days
DT50	0.201	0.121	0.235	0.826
DT90	0.668	0.402	0.78	2.74
100 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model	Single First Order (SFO)
Chi-square	19	12.3	18.5	6.59
DTX values in days
DT50	0.651	0.585	0.669	2.75
DT90	2.16	1.94	2.22	9.13

nd = not determinable

 

 

 

 

 

Validity criteria:

- reference substance to be degraded within the expected time interval (for aniline, usually less than 2 weeks)
- initial mass balance for non-labelled substance should be between 70% and 110%

Observed value:

- Mineralisation of the reference item > 50% after 6 days and >= 65% after 27 days both, in the reference control and the solvent control.
- Initial concentration for the non-labelled substance was between >91 and <=100%

Validity criteria fulfilled:

yes

Remarks:

A mass balance of ca. 100% could be established for day 20 and beyond.

Conclusions:

The simulation degradation testing (OECD TG 309) with river water (SPM 13 mg DW/L) performed at 12 °C over 65 days on cold test item TCD Alcohol DM had the following results: Transformation of TCD Alcohol DM started directly after application. Already after two days, the detected concentration was < 10 % of the applied concentration.
The TCD Alcohol DM isomers 1, 2 and 3 were first oxidized to an isomeric mixture of TCD Monoacids followed by further oxidation to the TCD Diacids under steady decrease of the TCD Monoacids. The concentration of the TCD Diacids remained in a plateau to the end of the study.
Apparent first order DT50 for TCD Alcohol DM: 0.121 d - 0.235 d (20 µg/L initial conc.); 0.585 d - 0.669 d (100 µg/L initial conc.).

Executive summary:

A reliable study on Aerobic Mineralisation in Surface Water according to OECD TG 309 (adopted April 2004) was performed (compliant with GLP) with the test item TCD Alcohol DM over a test period of 65 days at 12 °C. Natural surface water (field fresh sampled) of the river ‘LEINE’, containing a natural concentration of suspended matter of approx. 13 mg DW/L was used. The test item is a multi-constituent substance being constituted by > 95% (mass) of three structural isomers, namely TCD Alcohol DM1 (TCDM1, 36.78% mass), TCD Alcohol DM2 (TCDM2, 33.56% mass), and TCD Alcohol DM3 (TCDM1, 25.25% mass).
The test was performed with "cold" test item because different strategies of synthesis considered for 14C-labelling failed due to the required high pressure (4 specialized laboratories inquired but declined).
The lowest test item concentration of 20 µg/L test material was selected to assure an accurate kinetic analysis for the degradation kinetics of all three isomers present in the test item. Because the multi-constituent test item is constituted by ca. 1/3, each, of three main structural isomers, the concentration per isomer is ca. 6.7 µg/L. This agrees with the guideline recommendation of less than 10 µg/L for the lower concentration.
The test item was analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS, APCI positive). Concentrations were determined by an external standard calibration using the test item as standard.
The test item was rapidly oxidized in surface water by microorganisms as demonstrated by the preliminary study and confirmed by the definite study. Monocarboxylic and dicarboxylic acids were formed as metabolites.
Dicarboxylic acids (TCD Diacid 1, 2, and 3) were also analysed by LC-HRMS (ESI negative) using an authentic standard specifically synthesized. This standard was a mixture of the corresponding three tricyclic aliphatic dicarboxylic acids.
Monocarboxylic acids (TCD Monoacids) were analysed by a semi-quantitative assay with LC-HRMS (ESI negative) because no authentic standard was available.
The analytical methods used for the test item analysis and for the analysis of the TCD Diacids were validated prior to the start of the definite study based on the guideline SANTE/2020/12830, Rev.1.

Transformation of TCD Alcohol DM started directly after application. Already after two days the detected concentration was < 10 % of the applied concentration. The TCD Alcohol DM isomers 1, 2 and 3 were first oxidized to an isomeric mixture of TCD Monoacids followed by further oxidation to the TCD Diacids under steady decrease of the TCD Monoacids. The concentration of the TCD Diacids remained in a plateau to the end of the study. Based on the decline of parent TCD alcohol DM as well as the increase of TCD diacid (isomer specific identification and quantification), biological oxidation efficiency was independent of the specific structural isomer involved.
The deviation Δ of the experimentally determined accurate mass for the TCD monoacids was ≤ - 4.8 ppm in relation to the theoretical mass of the TCD monoacids indicating that these metabolites could be identified with sufficient quality.
In the sterile control, no transformation of TCD Alcohl DM was observed (recovery ca. 100%), confirming biological transformation.

The kinetic evaluations were done based on the FOCUS guidance document on estimating persistence and degradation kinetics. DTx values were calculated with single first order kinetics (SFO). The following results were obtained:

 

		20 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model:	Single	First	Order	(SFO)
Chi-square	1.03	nd	2.06	0.13
DTX	values	in	days
DT50	0.201	0.121	0.235	0.826
DT90	0.668	0.402	0.78	2.74
		100 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model:	Single	First	Order	(SFO)
Chi-square	19	12.3	18.5	6.59
DTX	values	in	days
DT50	0.651	0.585	0.669	2.75
DT90	2.16	1.94	2.22	9.13

 

All validity criteria of the OECD test guideline were met. A mass balance of ca. 100% could be established for day 20 and beyond.

 

Description of key information

The simulation degradation testing (OECD TG 309) with river water (SPM 13 mg DW/L) performed at 12 °C over 65 days on cold test item TCD Alcohol DM had the following results: Transformation of TCD Alcohol DM started directly after application. Already after two days, the detected concentration was < 10 % of the applied concentration.
The TCD Alcohol DM isomers 1, 2 and 3 were first oxidized to an isomeric mixture of TCD Monoacids followed by further oxidation to the TCD Diacids under steady decrease of the TCD Monoacids. The concentration of the TCD Diacids remained in a plateau to the end of the study.
Apparent first order DT50 for TCD Alcohol DM: 0.121 d - 0.235 d (20 µg/L initial conc.); 0.585 d - 0.669 d (100 µg/L initial conc.).

As a key value for chemical safety assessment, the largest value obtained for DT50 from testing with the lower initial concentration was selected, because the lower concentration range is regarded to be of higher relevance in relation to a) actual environmental exposure and b) apparent first order kinetics assumptions.

Key value for chemical safety assessment

Half-life in freshwater:

0.235 d

at the temperature of:

12 °C

Additional information

A reliable study on Aerobic Mineralisation in Surface Water according to OECD TG 309 (adopted April 2004) was performed (compliant with GLP) with the test item TCD Alcohol DM over a test period of 65 days at 12 °C. Natural surface water (field fresh sampled) of the river ‘LEINE’, containing a natural concentration of suspended matter of approx. 13 mg DW/L was used. The test item is a multi-constituent substance being constituted by > 95% (mass) of three structural isomers, namely TCD Alcohol DM1 (TCDM1, 36.78% mass), TCD Alcohol DM2 (TCDM2, 33.56% mass), and TCD Alcohol DM3 (TCDM1, 25.25% mass).
The test was performed with "cold" test item because different strategies of synthesis considered for 14C-labelling failed due to the required high pressure (4 specialized laboratories inquired but declined).
The lowest test item concentration of 20 µg/L test material was selected to assure an accurate kinetic analysis for the degradation kinetics of all three isomers present in the test item. Because the multi-constituent test item is constituted by ca. 1/3, each, of three main structural isomers, the concentration per isomer is ca. 6.7 µg/L. This agrees with the guideline recommendation of less than 10 µg/L for the lower concentration.
The test item was analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS, APCI positive). Concentrations were determined by an external standard calibration using the test item as standard.
The test item was rapidly oxidized in surface water by microorganisms as demonstrated by the preliminary study and confirmed by the definite study. Monocarboxylic and dicarboxylic acids were formed as metabolites.
Dicarboxylic acids (TCD Diacid 1, 2, and 3) were also analysed by LC-HRMS (ESI negative) using an authentic standard specifically synthesized. This standard was a mixture of the corresponding three tricyclic aliphatic dicarboxylic acids.
Monocarboxylic acids (TCD Monoacids) were analysed by a semi-quantitative assay with LC-HRMS (ESI negative) because no authentic standard was available.
The analytical methods used for the test item analysis and for the analysis of the TCD Diacids were validated prior to the start of the definite study based on the guideline SANTE/2020/12830, Rev.1.

Transformation of TCD Alcohol DM started directly after application. Already after two days the detected concentration was < 10 % of the applied concentration. The TCD Alcohol DM isomers 1, 2 and 3 were first oxidized to an isomeric mixture of TCD Monoacids followed by further oxidation to the TCD Diacids under steady decrease of the TCD Monoacids. The concentration of the TCD Diacids remained in a plateau to the end of the study. Based on the decline of parent TCD alcohol DM as well as the increase of TCD diacid (isomer specific identification and quantification), biological oxidation efficiency was independent of the specific structural isomer involved.
The deviation Δ of the experimentally determined accurate mass for the TCD monoacids was ≤ - 4.8 ppm in relation to the theoretical mass of the TCD monoacids indicating that these metabolites could be identified with sufficient quality.
In the sterile control, no transformation of TCD Alcohl DM was observed (recovery ca. 100%), confirming biological transformation.

The kinetic evaluations were done based on the FOCUS guidance document on estimating persistence and degradation kinetics. DTx values were calculated with single first order kinetics (SFO). The following results were obtained:

 

		20 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model:	Single	First	Order	(SFO)
Chi-square	1.03	nd	2.06	0.13
DTX	values	in	days
DT50	0.201	0.121	0.235	0.826
DT90	0.668	0.402	0.78	2.74
		100 µg/L
	TCDM1	TCDM2	TCDM3	TCD Monoacid
Model:	Single	First	Order	(SFO)
Chi-square	19	12.3	18.5	6.59
DTX	values	in	days
DT50	0.651	0.585	0.669	2.75
DT90	2.16	1.94	2.22	9.13

 

All validity criteria of the OECD test guideline were met. A mass balance of ca. 100% could be established for day 20 and beyond.

 

In accordance with column 2 of REACH Annex IX and X No 9.2, no further testing on sediment biotic degradation and identification of degradation products needs to be conducted as the CSA does not indicate the need for further testing: TCD Alcohol DM does not have a high potential for adsorption (log Kow < 3 IUCLID Sect. 4.7, low estimated Koc of 16.8, IUCLID Sect. 5.4.1). Based on the valid simulation test on degradation in surface water (OECD TG 309; Noack, 2021), TCD Alcohol DM is rapidly transformed to the dicarboxylic acid derivative. Any losses due to adsorption could reliably be excluded based on a mass balance of ca. 100 %. No relevant further information could be expected from additional simulation testing with sediment.