L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to Section 13.2 for read-across justification.

Reason / purpose for cross-reference:

read-across source

Test performance:

The procedural control, aniline, biodgraded 61% and 70% within 7 and 14 days respectively. As this is above 40 % and 65 %, for day 7 and 14, this test validity criterion was fulfilled. The inoculum blank had an oxygen demand <60 mg O2/L. The difference of extremes of the 3 replicates of the removal of the test item is less than 20 %. Therefore, in accordance with the OECD 301 guideline, the test is considered valid.

Key result

Parameter:

% degradation (O2 consumption)

Value:

60

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicates incubates

Key result

Parameter:

% degradation (DOC removal)

Value:

99

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicates analysis

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicate analysis

Details on results:

BOD method: After 28 days cultivation the BOD (mg) of three samples consisting of sludge and test substance was measured. Using BOD the percentage of biodegradation (%Degr.) of each sample and the mean percentage of biodegradation (mean %Degr.) was determined.
A comparatively low value of 60 % mean biodegradability was observed by the BOD method. The reason for the reduced BOD presumably was that some test substance was incorporated in the body of the microorganisms.

TOC method: After 28 days also the DOC (mgC/l) of the three samples consisting of sludge and test substance was measured. Using TOC apparatus, the percentage of biodegradation (%Degr.) was determined by using DOC (mg C/L) of each sample and the mean percentage of biodegradation (mean %Degr.) was also determined from the 3 replicates.

HPLC method: After 28 days also HPLC of the three samples consisting of sludge and test substance was performed. From the residual amount of test substance the percentage of biodegradation (%Degr.) of each sample and the mean percentage of biodegradation (mean %Degr.) was determined.

Additionally, the nitrification of the samples was determined by FIA. The average percentage production of the theoretical amount of converted product (NH4-N) of the three samples consisting of sludge and test substance was found to be 97%.

In order to know the final state of nitrogen, the analysis of ammonia state was performed in a flow injection analysing method. The average generation rate of ammonia was 97 %. In addition, the analyses of nitrous acid state and nitric acid state were performed in a similar manner, however there was no generation of nitrous acid or nitric acid in both cases. Therefore, TOD was calculated by assuming transformation of nitrogenous bases to ammonia.

 

The biodegradation of the test substance was calculated based on the following formulae:

 

BOD

 

% degradation = ((BOD – B) / TOD) X 100

 

where:

 

BOD = Biochemical oxygen demand of (Sludge + Test substance) base (Measured value) (mg)

B = Biochemical oxygen demand of sludge blank base (Measured value) (mg)

TOD = Theoretical oxygen demand required when test substance is completely oxidized (Calculated value) (mg) (assuming test item purity = 100 % and assuming the form of N as NH3)

 

TOC

 

% degradation = ((DOCw – DOCs) / DOCw) X 100

 

where:

 

DOCs = Residual amount of dissolved organic carbon in (Sludge + Test substance) base (Measured value) (mg C)

DOCw = Residual amount of dissolved organic carbon in (Water + Test substance) base (Measured value) (mg C)

 

HPLC

 

% degradation = ((Sw – Ss) / Sw) X 100

 

where:

 

Ss = Residual amount of test substance in (Sludge + Test substance) base (Measured value) (mg)

Sw = Residual amount of test substance in (Water + Test substance) base (Measured value) (mg)

 

Table 1       Results of test liquid analysis

 

		Abiotic control	Test Replicates			Theoretical amount
			1	2	3	(mg)
BOD	(mg)	1.8	17.1	18.3	19.0	30.3
DOC residual	(mg C)	12.0	0.2	0.0	0.0	12.4
	(%)^*	97	2	0	0
Test substance residual (HPLC)	(mg)	29.9	0.0	0.0	0.0	30
	(%)^*	100	0	0	0
Ammonia state nitrogen generation (FIA)	(mg N)	0.3	9.6	8.8	9.4	9.6 (mgN)
	(%)^*	3	100	92	98

 * calculated from the following equations;

 

Residual rate (%) = (Residual amount mg / Theoretical amount mg) X 100

Generation rate (%) = (Generation amount mg / Theoretical amount mg) X 100

 

Table 2       Biodegradation results after 28 days

 

	Rate of biodegradation (%)
	Rep 1	Rep 2	Rep 3
Result by BOD (NH3)	56	60	63
Result by TOC	98	100	100
Result by HLPC	100	100	100

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The substance was reported to be 60, 99 and 100 % degraded by Day 28 using BOD, TOC and HPLC analytical techniques. The substance is readily biodegradable.

Executive summary:

OECD 301C (1993) - A ready biodegradability test was performed in accordance with OECD Test Guideline 301C (Modified MITI Test; 1992) and the Environmental Protection Agency No. 5, Pharmaceutical Affairs Bureau No. 615, 49 Basic Industries Bureau No. 392, on July 13, 1974.

 

The test item (concentration 100 mg/L) was added to three test vessels containing mineral medium inoculated with activated sludge (30 mg/L concentration of suspended solids) from a cultivated laboratory culture.  The inoculum originated from 10 different sites with the following origins: sewage treatment plant, surface waters (river and lake) and overlying beach surface waters in contact with the atmosphere. One abiotic control, one inoculum blank and one reference control were run concurrently. Aniline was used as the reference substance.

 

A closed system oxygen consumption measurer was used to monitor BOD of the test solutions continuously during the incubation period of 28 days.

 

At the end of the incubation period, the following analyses were conducted for the test solutions and control blank:

- Determination of DOC (using a TOC analyser);

- Determination of the test item concentration in test solution by HPLC;

- Measurement of pH.

 

The mean percentage biodegradation on Day 28 was 60 % by BOD. DOC analysis indicated that the test item had gone through ultimate degradation, with 100 % removal after 28 days.  concluded that the test item had fully degraded. Three replicate HPLC analyses indicated that 100 % of the applied test item had degraded, showing the test item had undergone full primary degradation, supporting the conclusion of completer test item removal and the ultimate degradations seen in the BOD (60 %) and DOC (100 %) measures.  The pH was adjusted from 8.1 to 7.0 at the start of the test in all BOD test vessels. In all measured BOD vessels the pH ranged from 8.7-8.9 at the end of the test period. Although test solution pH was not within pH 6.0-8.5 at the end of the test the oxygen consumption of the test item was ≥ 60 % at Day 28, therefore repeat testing at a lower test item concentration was not required as stipulated in the OECD 301C Guideline (Paragraph 19, 1992).

The procedural (reference) control, aniline, biodgraded 61% and 70% within 7 and 14 days respectively. As this is above 40 % and 65 %, for day 7 and 14, this test validity criterion was fulfilled. The inoculum blank had an oxygen demand <60 mg O2/L. The difference of extremes of the 3 replicates of the removal of the test item is less than 20 %.  Therefore, in accordance with the OECD 301 guideline, all validity criteria for this test were fulfilled.

With an average 28 d biodegradation value of 60 % and 99 % as per BOD and DOC analysis, the test item can be considered to be readily biodegradable under the conditions of this test. Based on DOC the test item is considered to have undergone full ultimate degradation (i.e. tranformed full to water and CO2).

The difference in the BOD and DOC measures is possibly due to the incorporation/use of arginine in the microorganisms, as it is an essential amino acid to prokaryotes and an energy source for many microorganisms.  As the microorganisms and solids are separated by centrifugation before DOC analysis, this DOC would not be present.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May - 30 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with GLP and OECD Guideline 301C.

Justification for type of information:

Please refer to the read-across justification attached in Section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Version / remarks:

17 July 1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal wastewater (returned sewage sludge from STP) and "river, lake and sea water" (surface water and surface soil of beach which is contact with atmosphere).
- Laboratory culture: Yes, after collection
- Method of cultivation: 500 mL of sludge filtrate was used from the varying sample sites, and 5 L of old activated sludge filtrate were mixed to obtain 10 L. The pH was adjusted to 7.0 ± 1.0, and the mixture was aerated (outdoor air passed through a prefilter was used for aeration) in a cultivation tank. After stopping the aeration for about 30 minutes, the supernatant was removed, this was about ⅓ of total amount (i.e. 3.3 L). An equal amount of dechlorinated water (ca. 3.3 L) was added and the mixture was again aerated. Sewage (glucose, peptone and monopotassium phosphate dissolved in dechlorinated water so as to make the content of each substance 5 (W/V)% with pH adjusted to 7.0 ± 1.0 using sodium hydroxide was used for sewage) was added so as to make the concentration of supernatant-exchanged liquid 0.1%. This operation was repeated once a day, and the liquid was cultivated to obtain the activated sludge. The cultivation temperature was 25°C ± 2°C.

- Storage conditions: Not reported
- Storage length: From 13 April - 14 May 1993
- Preparation of inoculum for exposure: See "method of cultivation".
- Pretreatment: No.
- Concentration of sludge: Suspended solids concentration of the activated sludge was 4800 mg/L. The prepared activated sludge was added into the test system so as to obtain a suspended solids concentration of 30 mg/L.
- Water filtered: Not reported.
- Type and size of filter used, if any: Not reported.

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:

DOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: 3 mL of each solution A, B, C, and D stipulated in the factory wastewater testing method, biochemical oxygen consumption (21. of JIS K 0102-1986) (assumed to be similar to, or equivalent to solutions A,B, C and D of the OECD 301C guideline, Paragraph 5, 1992), were diluted with purified water to obtain a solution of 1 L by mixing, and pH was adjusted to 7.0.
- Additional substrate: No.
- Solubilising agent (type and concentration if used): No.
- Test temperature: 25±1ºC
- pH: Mineral medium and sewage innoculum adjusted to pH 7.
- pH adjusted: yes
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: Activated sludge was inoculated to test solutions so as to obtain a concentration of suspended solids of 30 mg/L.
- Continuous darkness: Not reported
- Other: -

TEST SYSTEM
- Culturing apparatus: 300 mL cultivation bottle
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: Not reported
- Method used to create anaerobic conditions: Not reported
- Measuring equipment: Closed system oxygen consumption measuring instrument (Curo Meter manufactured by Okura Electric) (Data Processor manufactured by Asahi Gauge Manufacturing).
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Soda lime traps.
- Other: -

SAMPLING
- Sampling frequency: BOD continuously monitored by equipment. Test item and DOC analysis conducted at Day 28.
- Sampling method: BOD continuosly monitored by equipment. For HPLC and TOC analysis, subsamples (10 mL) of pre-treated test solution were taken from replicate flasks and prepared for analysis.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not reported.
- Other: -

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: No
- Other: Aniline reference control.

STATISTICAL METHODS: Not required

Reference substance:

aniline

Test performance:

Since the degrees of decomposition of aniline after 7 and 14 days obtained from BOD were 61% and 70%, respectively, the test condition of this test was recognized to be effective.

Key result

Parameter:

% degradation (O2 consumption)

Value:

60

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicates

Key result

Parameter:

% degradation (DOC removal)

Value:

99

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicates

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Remarks on result:

other: Mean of 3 replicates

Details on results:

BOD method: After 28 days cultivation the BOD (mg) of three samples consisting of sludge and test substance was measured. Using BOD the percentage of biodegradation (%Degr.) of each sample and the mean percentage of biodegradation (mean %Degr.) was determined.
A comparatively low value of 60% mean biodegradability was observed by the BOD method. The reason for the reduced BOD presumably was that some test substance was incorporated in the body of the microorganisms.

TOC method: After 28 days also the TOC (mgC/l) of the three samples consisting of sludge and test substance was measured. Using TOC and DOC the percentage of biodegradation (%Degr.) of each sample and the mean percentage of biodegradation (mean %Degr.) was determined.

HPLC method: After 28 days also HPLC of the three samples consisting of sludge and test substance was performed. From the residual amount of test substance the percentage of biodegradation (%Degr.) of each sample and the mean percentage of biodegradation (mean %Degr.) was determined.

Additionally, the nitrification of the samples was determined by FIA. The average percentage production of the theoretical amount of converted product (NH4-N) of the three samples consisting of sludge and test substance was found to be 97%.

In order to know the final state of nitrogen, the analysis of ammonia state nitrogen was performed in the flow injection analyzing method. As a result, the average generation rate was 97 %. In addition, the analyses of nitrous acid state nitrogen and nitric acid state nitrogen were performed in the similar way without recognizing the generation in both cases. Therefore, TOD was calculated by assuming the state of nitrogen as that of ammonia.

 

The degree of decomposition of test substance was calculated based on the following formulae;

 

BOD

 

% degradation = ((BOD – B) / TOD) X 100

 

where:

 

BOD = Biochemical oxygen demand of (Sludge + Test substance) base (Measured value) (mg)

B = Biochemical oxygen demand of sludge blank base (Measured value) (mg)

TOD = Theoretical oxygen demand required when test substance is completely oxidized (Calculated value) (mg) (assuming test item purity = 100 % and assuming the form of N as NH3)

 

TOC

 

% degradation = ((DOCw – DOCs) / DOCw) X 100

 

where:

 

DOCs = Residual amount of dissolved organic carbon in (Sludge + Test substance) base (Measured value) (mg C)

DOCw = Residual amount of dissolved organic carbon in (Water + Test substance) base (Measured value) (mg C)

 

HPLC

 

% degradation = ((Sw – Ss) / Sw) X 100

 

where:

 

Ss = Residual amount of test substance in (Sludge + Test substance) base (Measured value) (mg)

Sw = Residual amount of test substance in (Water + Test substance) base (Measured value) (mg)

 

Table 1       Results of test liquid analysis

 

		Abiotic control	Test Replicates			Theoretical amount
			1	2	3	(mg)
BOD	(mg)	1.8	17.1	18.3	19.0	30.3
DOC residual	(mg C)	12.0	0.2	0.0	0.0	12.4
	(%)^*	97	2	0	0
Test substance residual (HPLC)	(mg)	29.9	0.0	0.0	0.0	30
	(%)^*	100	0	0	0
Ammonia state nitrogen generation (FIA)	(mg N)	0.3	9.6	8.8	9.4	9.6 (mgN)
	(%)^*	3	100	92	98

 * calculated from the following equations;

 

Residual rate (%) = (Residual amount mg / Theoretical amount mg) X 100

Generation rate (%) = (Generation amount mg / Theoretical amount mg) X 100

 

Table 2       Biodegradation results after 28 days

 

	Rate of biodegradation (%)
	Rep 1	Rep 2	Rep 3
Result by BOD (NH3)	56	60	63
Result by TOC	98	100	100
Result by HLPC	100	100	100

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The substance was reported to be 60, 99 and 100 % degraded by Day 28 using BOD, TOC and HPLC analytical techniques. The substance is readily biodegradable.

Executive summary:

OECD 301C (1993) - A ready biodegradability test was performed in accordance with OECD Test Guideline 301C (Modified MITI Test; 1992) and the Environmental Protection Agency No. 5, Pharmaceutical Affairs Bureau No. 615, 49 Basic Industries Bureau No. 392, on July 13, 1974.

 

The test item (concentration 100 mg/L) was added to three test vessels containing mineral medium inoculated with activated sludge (30 mg/L concentration of suspended solids) from a cultivated laboratory culture. The inoculum originated from a sewage treatment plant and overlying beach surface waters and soil. One abiotic control, one inoculum blank and one reference control were run concurrently. Aniline was used as the reference substance.

 

A closed system oxygen consumption measurer was used to monitor BOD of the test solutions continuously during the incubation period of 28 days.

 

At the end of the incubation period, the following analyses were conducted for the test solutions and control blank:

- Determination of DOC (using a TOC analyser);

- Determination of the test item concentration in test solution by HPLC;

- Measurement of pH.

 

The mean percentage biodegradation on Day 28 was 60 % by BOD. Three replicate HPLC analyses indicated that 100 % of the applied test item had degraded, which supports the fact the test item was fully degraded. DOC analysis also concluded that the test item had fully degraded. The pH in all measured BOD vessels was in the range of 8.1-8.9 through the test period. Although test solution pH was out with the 6.5-8.5 range stipulated in the OECD 301C Guideline, it did not negatively impact the rate of biodegradation (i.e. biodegradation was ≥60 % at Day 28) and therefore repeat testing was not required at a lower test item concentration.

 

In the reference test solution containing aniline, the percentage biodegradation by BOD was 61 and 70 % on Days 7 and 14, respectively. As per the OECD 301C Guideline, testing proficiency can be demonstrated when the percent degradation of aniline exceeds 40 % at Day 7 and 65 % at Day 14. In this experiment the reference item met the validity criteria.

With an average 28 d biodegradation value of 60 % (BOD), the test item can be considered to be readily biodegradable under the conditions of this test.

 

All validity criteria for this test were fulfilled as described above.

Description of key information

Readily biodegradable: 28-day biodegradation (based on BOD) (read-across) = 60 %; OECD 301C; Anon. (1993)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

OECD 301C (1993) - A ready biodegradability test was performed in accordance with OECD Test Guideline 301C (Modified MITI Test; 1992) and the Environmental Protection Agency No. 5, Pharmaceutical Affairs Bureau No. 615, 49 Basic Industries Bureau No. 392, on July 13, 1974.

 

The test item (concentration 100 mg/L) was added to three test vessels containing mineral medium inoculated with activated sludge (30 mg/L concentration of suspended solids) from a cultivated laboratory culture. The inoculum originated from a sewage treatment plant and overlying beach surface waters and soil. One abiotic control, one inoculum blank and one reference control were run concurrently. Aniline was used as the reference substance.

 

A closed system oxygen consumption measurer was used to monitor BOD of the test solutions continuously during the incubation period of 28 days.

 

At the end of the incubation period, the following analyses were conducted for the test solutions and control blank:

- Determination of DOC (using a TOC analyser);

- Determination of the test item concentration in test solution by HPLC;

- Measurement of pH.

 

The mean percentage biodegradation on Day 28 was 60 % by BOD. Three replicate HPLC analyses indicated that 100 % of the applied test item had degraded, which supports the fact the test item was fully degraded. DOC analysis also concluded that the test item had fully degraded. The pH in all measured BOD vessels was in the range of 8.1-8.9 through the test period. Although test solution pH was out with the 6.5-8.5 range stipulated in the OECD 301C Guideline, it did not negatively impact the rate of biodegradation (i.e. biodegradation was ≥60 % at Day 28) and therefore repeat testing was not required at a lower test item concentration.

 

In the reference test solution containing aniline, the percentage biodegradation by BOD was 61 and 70 % on Days 7 and 14, respectively. As per the OECD 301C Guideline, testing proficiency can be demonstrated when the percent degradation of aniline exceeds 40 % at Day 7 and 65 % at Day 14. In this experiment the reference item met the validity criteria.

 

With an average 28 d biodegradation value of 60 % (BOD), the test item can be considered to be readily biodegradable under the conditions of this test.

 

All validity criteria for this test were fulfilled as described above.