Fatty acid chlorides, C12-18 (even numbered) and C18 unsatd., reaction products with sodium N-methyltaurinate

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-23 to 2013-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acid chlorides, C12-18 (even numbered) and C18 unsatd., reaction products with sodium N-methyltaurinate

EC Number:

939-529-5

Cas Number:

1471313-51-5

Molecular formula:

not applicable

IUPAC Name:

Fatty acid chlorides, C12-18 (even numbered) and C18 unsatd., reaction products with sodium N-methyltaurinate

Details on test material:

- Name of test material (as cited in study report): Sodium methyl cocoyl taurate
- Physical state: Slightly yellowish coarse powder, solid
- Stability under test conditions: stable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.500 – 1.00 – 2.00 – 4.00 – 8.00 mg/L
- Sampling method: All concentration levels of the test item and the control were analysed by LC-MS/MS at the start of the exposure intervals on days 7, 9, 14 (0 h) and at the end of the exposure intervals on days 9, 12, 16 (48 or 72 h).
For the analyses of the old media, separate replicates without daphnids and algae were prepared and stored under test conditions until sampling.
- Sample storage conditions before analysis: All original samples were stored room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (8.00 mg/L of the test item were weighed out) was freshly prepared with dilution water at the start of the test period and at every water renewal day.Dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 0.500 – 1.00 – 2.00 – 4.00 – 8.00 mg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: renewals 3 times per week


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-02-06 2013-02-08 2013-02-15 2013-02-18 2013-02-20 2013-02-22
8.00 173 171 178 155 166 146
Control 162 164 178 171 169 148

Test temperature:

Temperature [°C]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-02-06 2013-02-08 2013-02-15 2013-02-18 2013-02-20 2013-02-22
8.00 21.2 20.6 19.9 20.3 21.0 20.9
Control 19.7 20.5 19.6 20.3 20.2 21.0

pH:

pH-Values
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-02-06 2013-02-08 2013-02-15 2013-02-18 2013-02-20 2013-02-22
8.00 7.81 7.60 8.74 7.78 7.82 7.51
Control 8.14 7.66 8.89 7.84 8.34 7.64

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-02-06 2013-02-08 2013-02-15 2013-02-18 2013-02-20 2013-02-22
8.00 8.59 7.16 8.69 7.82 8.90 6.76
Control 8.75 6.75 10.29 7.88 8.96 7.85

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 mg CaCO3/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
Based on the results of an acute immobilisation test (NOACK Lab-ID: 120425CS/DAI15129) conducted at the test facility, the definitive reproduction test was carried out with 5 concentration levels of the test item in the range of 0.500 to 8.00 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

is UVCB means 100% act.ingr.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
The test item did not induced biologically significant adult mortality of > 20 % at any of the tested concentration levels during the test period of 21 days.
At the concentration level of 8.00 mg/L, the adult mortality was 10 %. At the concentration levels of 0.500 to 4.00 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. The EC10 for the adult mortality was deduced directly from the observation data to be 8.00 mg/L. An EC50 could not be determined, because adult mortality of > 50 % was not observed.
- No. of offspring produced per day per female:
The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 93 in the control group. The reproductive output was statistically significant increased in comparison to the control at the concentration level of 8.00 mg/L (One Way Analysis of Variance, DUNNETT’S method, p = 0.05). At the concentration level of 8.00 mg/L, an increased number of living juveniles per brood were observed and a reduced number of broods at two of nine surviving daphnids. Furthernore, the calculated coefficient of variation of the number of living offspring of 29 % for the concentration level 8.00 mg/L was biologically noticeable increased.
At the concentration levels of 0.500 to 4.00 mg/L, the reproductive output was comparable to the reproductive output at the control.
The EC10 for the increase of the reproductive output was assed to be within the range of 4.00 to 8.00 mg/L. An EC50 could not be calculated, because the increase of the reproductive output was limited to a maximum of 33 %.
- Body length and weight of parent animals:
The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.500 to 8.00 mg/L were determined to be in the range of 5.28 to 5.60 mm per daphnid, 5.45 mm per daphnid in the control group.
The mean dry body weight was determined to be in the range of 1.21 to 1.42 mg per daphnid in the concentration levels 0.500 to 8.00 mg/L and 1.49 mg per daphnid in the contro
- Type and number of morphological / behavioural abnormalities:
please refer to the respective table
- Time to first brood release or time to hatch:
The first brood was released between days 8 and 10 by all parental daphnids of the control and the concentration levels of 0.500 to 4.00 mg/L. At the concentration level of 8.00 mg/L, one of nine surviving daphnids released the first brood noticeably delayed on day 18.
Four broods were produced by the surviving parental daphnids at the tested concentration levels of 0.500 to 4.00 mg/L and in the control. At the concentration level of 8.00 mg/L, two of nine surviving daphnids released less than four broods

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.69 (CI 1.41 - 2.12) mg/L

Reported statistics and error estimates:

The NOEC and the LOEC, based on the increase of the reproductive output, were deduced directly from the results. Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test and analysis of variance (parametric ANOVA, DUNNETT’S method).
Prior to running the parametric ANOVA, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests should be 0.05. Actually, the P-value was < 0.05 and therefore the normality test failed for the statistical evaluation of the reproduction rates and the intrinsic rates of natural increase. Furthermore, the equal variance test for the evaluation of the reproduction rates failed (P value < 0.05).
The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
For the determination of significant deviations of the reproduction rates and the intrinsic rates of natural increase, One Way Analysis of Variance, DUNNETT’S method (p = 0.05) was used.
The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The EC10-values for the increase of the reproductive output and the adult mortality after 21 days were deduced directly from the observation data without mathematical calculation.
The EC50-value for the reference item was calculated by sigmoidal dose-response regression. The 95 % confidence limits for the EC-values were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.
An EC50-value for the increase of the reproductive output or the adult mortality after 21 days could not be calculated because effects > 50 % were not observed.

Any other information on results incl. tables

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal test item conc. [mg/L]												No. of parents prod. juv.	Mean no. of Juveniles per parent producing juveniles			Comparison versus control
	Number of living juveniles in replicate no.										Total no.				CV
																Inc.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
8.00	141	149	140	92	45	124	167	137	119	--	1114	9	124 ±	36	29*	33	yes
4.00	101	114	102	86	79	84	82	85	97	92	922	10	92 ±	11	12	-1	no
2.00	89	111	101	115	104	101	119	93	93	93	1019	10	102 ±	10	10	10	no
1.00	77	111	99	87	98	101	76	85	107	91	932	10	93 ±	12	13	1	no
0.500	95	108	97	92	92	98	95	82	87	93	939	10	94 ±	7	7	1	no
Control	80	89	103	90	94	103	85	89	103	93	929	10	93 ±	8	9	—	—

First Appearance of Living Juveniles in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles										First	Mean
	in replicate no.										appearance	number
	1	2	3	4	5	6	7	8	9	10	mean day	of broods
8.00	8	9	8	8	18^1)	9	8	8	9	--	9.4	3.56^2)
4.00	8	8	9	8	8	8	8	9	8	9	8.3	4.00
2.00	9	8	9	9	8	9	9	8	8	9	8.6	4.00
1.00	10	8	9	8	9	8	8	8	9	8	8.5	4.00
0.500	8	9	9	9	8	9	9	9	9	8	8.7	4.00
Control	8	8	8	8	8	8	9	8	9	8	8.2	4.00

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal test item concentration [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
8.00	45	1114	1159	4
4.00	55	922	977	6
2.00	48	1019	1067	5
1.00	10	932	942	1
0.500	5	939	944	1
Control	0	929	929	0

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
8.00	10	10	10
4.00	0	0	0
2.00	0	0	0
1.00	0	0	0
0.500	0	0	0
Control	0	0	0

Total Body Length of the Parental Daphnids

Nominal test item concentration [mg/L]	Total length of the parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
8.00	5.00	5.75	5.50	5.50	5.25	5.00	5.50	5.75	5.50	--	5.42	9	10.9	1.21
4.00	5.25	5.25	5.00	5.25	5.50	5.50	5.25	5.75	5.50	5.50	5.38	10	13.6	1.36
2.00	5.75	5.75	5.50	5.50	6.00	5.50	5.50	5.75	5.75	5.00	5.60	10	14.2	1.42
1.00	5.25	5.00	5.50	5.25	5.00	5.25	5.50	5.50	5.25	5.25	5.28	10	13.4	1.34
0.500	5.50	5.00	5.50	5.25	5.25	5.25	5.25	5.25	5.75	5.00	5.30	10	13.9	1.39
Control	5.75	5.25	5.25	5.50	5.50	5.75	5.50	5.25	5.50	5.25	5.45	10	14.9	1.49

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The statistically significant increase of the reproductive output at the concentration level of 8.00 mg/L and the noticeably increased coefficient of variation of the number of living offspring were the most sensitive effects in this study. At the concentration level of 8.00 mg/L, an increased number of living juveniles per brood was observed.
Based on the significant increase of the reproductive output and the noticeably increased coefficient of variation of the number of living offspring at the concentration level of 8.00 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 4.00 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 8.00 mg/L.
Based on the nominal concentrations of the active ingredient, the NOEC was 2.89 mg a.i./L and the LOEC was 5.78 mg a.i./L.
The EC10 for the increase of the reproductive output was assed to be within the range of 4.00 to 8.00 mg/L, which corresponds to 2.89 – 5.78 mg a.i./L. An EC50 could not be calculated, because the increase of the reproductive output was limited to a maximum of 33 %.
At the concentration level of 8.00 mg/L, the adult mortality was 10 %. Therefore, the EC10 for the adult mortality was assessed at 8.00 mg/L, which corresponds to 5.78 mg a.i./L. An EC50 could not be determined, because adult mortality of > 50 % was not observed.
Endpoints assessed Sodium methyl cocoyl taurate
Nominal concentration
of the test item
[mg/L] Nominal concentration
of the active ingredient
[mg a.i./L]
EC10 Reproduction 4.00 mg/L < EC10 < 8.00 2.89 mg/L < EC10 < 5.78
EC10 Adult Mortality 8.00 5.78
NOECReproduction 4.00 2.89
LOECReproduction 8.00 5.78

Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 d) of the test item Sodium methyl cocoyl taurate (batch number: DEB4003323) was conducted according to OECD 211 (2008) from 2013-01-23 to 2013-03-01, with the definitive exposure phase from 2013-02-06 to     2013-02-27, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions with renewal of the test solutions three times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of Sodium methyl cocoyl taurate were selected as follows:  0.500 – 1.00 – 2.00 – 4.00 – 8.00 mg/L. This corresponds to the nominal concentrations of the active ingredient of 0.362 – 0.723 – 1.45 – 2.89 – 5.78 mg a.i./L based on the nominal content of 72.3 % (w/w).

The concentrations of the test item Sodium methyl cocoyl taurate were analytically verified by LC-MS/MS of samples taken at the start of the exposure intervals on days 7, 9, 14 (0 h) and at the end of the exposure intervals on days 9, 12, 16 (48 or 72 h) of all concentration levels and the control. The main component of the active ingredient (C12 part) was analytically verified. Details of the analytical method are presented in part 11.

The measured concentrations of the active ingredient at the start of the exposure intervals were in the range of 97 to 117 % of the nominal values. At the end of the exposure intervals (48 or 72 h), the measured concentrations of the active ingredient were in the range of 92 to 108 % of the nominal values. This indicates, that the test item concentrations were successfully maintained for the duration of the test.

The endpoints, based on the nominal concentrations of the test item and the active ingredient, are presented in Table 1.

·   The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 93 in the control group. The reproductive output was statistically significant increased in comparison to the control at the concentration level of 8.00 mg/L (One Way Analysis of Variance, Dunnett’smethod, p = 0.05). At the concentration level of 8.00 mg/L, an increased number of living juveniles per brood were observed and a reduced number of broods at two of nine surviving daphnids. Furthernore, the calculated coefficient of variation of the number of living offspring of 29 % for the concentration level 8.00 mg/L was biologically noticeable increased.
At the concentration levels of 0.500 to 4.00 mg/L, the reproductive output was comparable to the reproductive output at the control.
The EC₁₀ for the increase of the reproductive output was assed to be within the range of 4.00 to 8.00 mg/L. An EC₅₀ could not be calculated, because the increase of the reproductive output was limited to a maximum of 33 %.

·   The coefficient of variation of the number of living offspring produced per parent was 9 % in the control. At the concentration levels of 0.500 to 4.00 mg/L, the coefficient of variation was in the range of 7 to 13%. The calculated coefficient of variation of 29 % for the concentration level 8.00 mg/L was biologically noticeable increased. A coefficient of variation above 25 % is regarded to be biologically noticeable

·   Based on the significant increase of the reproductive output and the noticeably increased coefficient of variation of the number of living offspring at the concentration level of 8.00 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 4.00 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 8.00 mg/L.

·   The first brood was released between days 8 and 10 by all parental daphnids of the control and the concentration levels of 0.500 to 4.00 mg/L. At the concentration level of 8.00 mg/L, one of nine surviving daphnids released the first brood noticeably delayed on day 18.
Four broods were produced by the surviving parental daphnids at the tested concentration levels of 0.500 to 4.00 mg/L and in the control. At the concentration level of 8.00 mg/L, two of nine surviving daphnids released less than four broods

·   The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of natural increase determined for the surviving daphnids at the concentration levels of 0.500 to 8.00 mg/L were comparable to the IR at the control (One Way Analysis of Variance, Dunnett’s method, p = 0.05).

·   No stillborn juveniles or aborted egg were observed in the control group during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was in the range of 1 to 6 % in the tested concentration levels of 0.500 to 8.00 mg/L.

·   The test item did not induced biologically significant adult mortality of > 20 % at any of the tested concentration levels during the test period of 21 days.At the concentration level of 8.00 mg/L, the adult mortality was 10 %. At the concentration levels of 0.500 to 4.00 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. The EC₁₀for the adult mortality was deduced directly from the observation data to be 8.00 mg/L. An EC₅₀ could not be determined, because adult mortality of > 50 % was not observed.

·   The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.500 to 8.00 mg/L were determined to be in the range of 5.28 to 5.60 mm per daphnid, 5.45 mm per daphnid in the control group.

     The mean dry body weight was determined to be in the range of 1.21 to 1.42 mg per daphnid in the concentration levels 0.500 to 8.00 mg/L and 1.49 mg per daphnid in the control

·       No males or ephippia (winter eggs) were observed in the control or in the test groups.

 

·   Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

IMPORTANT NOTE TO AVOID CONFUSION:

As the registration substance is a UVCB this means that the test item is 100% active ingredient.

A summary of all test item related effects and the assessed endpoints based on the nominal concentrations of the test item is given in Table 1.

Table 1:           Test Item Related Effects on the Reproduction (NOEC, LOEC and EC₁₀)

                         (based on the nominal concentrations of the test item )

Effects		Sodium methyl cocoyl taurate Nominal concentrations of the test item [mg/L]
		Control		0.500 0.362		1.00 0.723		2.00 1.45		4.00 2.89		8.00 5.78
Mean Number of Living Juveniles per Producing Parent (Reproduction Rate ± SD)		93 ± 8		94 ± 7		93 ± 12		102 ± 10		92 ± 11		124 ± 36^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]		9		7		13		10		12		29^2)
Mean Number of Broods		4.00		4.00		4.00		4.00		4.00		3.56^3)
Appearance of First Brood [Mean Day]		8.2		8.7		8.5		8.6		8.3		9.4^4)
Mean Intrinsic Rates of Natural Increase		0.47		0.46		0.46		0.47		0.47		0.46
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]		0		1		1		5		6		4
Parental Daphnids: Mean Dry Weight [mg]		1.49		1.39		1.34		1.42		1.36		1.21
Parental Daphnids: Mean Body Length [mm]		5.45		5.30		5.28		5.60		5.38		5.42
Adult Mortality after 21 Days [%]		0		0		0		0		0		10
EC10 Reproduction		4.00 mg/L EC10 Reproduction 8.00 mg/L
EC10 Adult mortality		8.00 mg/L
NOECReproduction		4.00 mg/L
LOECReproduction		8.00 mg/L

SD        = standard deviation

¹⁾           = statistical significance (One Way Analysis of Variance, Dunnett’smethod, p = 0.05)

²⁾           = biologically noticeable coefficient of variation (> 25 %)

³⁾           = reduced number of broods at two of nine surviving daphnids (less than 4 broods)

⁴⁾           = delayed appearance of the 1^st brood on day 18 at one of nine surviving daphnids

a.i.        = active ingredient

 

 

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to DIN 38412 L 11 is monthly carried out with potassium dichromate as reference item.
The EC₅₀ of the reference item was 1.69 mg/L (95% confidence limits: 1.41 - 2.12 mg/L) after 24 hours and therefore within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.