C16-(branched), C20-(branched) and C24-(branched)-alkanes

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-10 to 2010-03-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Saturated solution and control
- Sampling method: The saturated solution of the test item and the control were analytically verified in the fresh media (0 h) and old media (48 or 72 h) via GC-FID at least once every 7days.
For the fresh media samples of the freshly prepared test solutions were taken and analysed. For the old media additionally prepared replicates with test item but without test animals were prepared and incubated under test conditions until analysis.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution of the test item with a nominal loading of 100 mg/L was prepared with dilution water two days prior to application. The test item was placed on the surface of the dilution water. A slow stirring procedure in a closed bottle was applied. Gentle stirring (to avoid the formation of an emulsion) was carried out for at least 48 h.
- Eluate: Dilution water
- Differential loading: Due to the sponsor’s request the definitive reproduction test was carried out with the saturated solution of the test item with a nominal loading of 100 mg/L as a limit test.
- Controls: 10 replicates without test item are included in every test series.

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: daily


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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal Loading
of the
Saturated Solution
[mg test item/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
100 189 173 173 168 183 183
Control 180 175 172 177 167 185

Test temperature:

Temperature [°C]
Nominal Loading
of the
Saturated Solution
[mg test item/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
100 21.7 21.4 21.5 21.7 21.9 22.0
Control 19.8 21.8 21.0 21.7 20.2 21.7

pH:

pH-Values
Nominal Loading
of the
Saturated Solution
[mg test item/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
100 7.58 7.67 7.48 7.34 7.54 7.40
Control 7.58 7.69 7.94 7.37 7.97 7.37

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal Loading
of the
Saturated Solution
[mg test item/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
100 8.08 8.37 7.90 6.58 8.26 7.92
Control 8.72 8.48 8.56 7.09 8.80 8.17

Salinity:

Not measured

Nominal and measured concentrations:

Please refer to " Any other information on materials and methods incl. tables"

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 (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, modified to a total hardness of 160 - 180 mg CaCO3/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: once per week in fresh and old media


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
- Test concentrations: 5 - 10 - 100 mg/L
- Results used to determine the conditions for the definitive study:
Immobilization Rates in the Preliminary Test after 24 and 48 h of Exposure
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal Loading
of the Saturated Solution

[mg test item/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
100 0 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L (saturated solution)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals: No immobilization or mortality of parent animals was observed, either in the control or in the saturated solution
- No. of offspring produced per day per female: In the control group and the saturated solution four to five broods were released during the test period by all of the daphnids.
The mean number of offspring alive produced per parent animal surviving at the end of the test was 111 juveniles in the control group and 107 juveniles in the saturated solution
- Body length and weight of parent animals: Please refer to the respective table.
- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males were observed in either the control or the test group during the test.
- Time to first brood release or time to hatch: The first brood was released by all parental daphnids of the control and the saturated solution between day 8 and 10

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.44 - 1.84) mg/L

Reported statistics and error estimates:

The NOEL and LOEL were determined directly from the results.
Significant deviations were determined in comparison to the control group using statistical standard procedures as normality test, equal variance test; t-test and MANN-WHITNEY Rank Sum Test.
t-test (p = 0.05) was used for the determination of the significant deviations for the number of juveniles. Statistical evaluation of the intrinsic rates of natural increase was carried out by using MANN-WHITNEY Rank Sum Test because normality test failed. The coefficients of variation around the mean number of living offspring produced per parent for the control and the saturated solution were evaluated.

Number of Juveniles in the Control and Test Group after 21 d

Nom. Load. of the Sat. Sol. [mg/L]																					No.	Mean No. of Juveniles			Compared
	Number of Juveniles in Replicate No.																			Total	of			CV	versus
																				No.	Parents	per Parent			Control
																					prod. Juv.	producing Juveniles			Red.
	1	2		3		4		5		6		7		8		9		10		å	N	MV ± SD		[%]	[%]
100	114	106		117		115		116		106		104		90		102		101		1071	10	107 ±	8.5	8	4
Control	125	103		105		122		129		96		92		124		113		103		1112	10	111 ±	13.2	12	--

Nom. Load. of the Sat. Sol.        = Nominal Loading of the Saturated Solution of theTest Item

N                                                   = Number of parents producing juveniles alive

MV ± SD                                        = Mean value ± Standard deviation

CV                                                 = Coefficient of variation

Red.                                              = Reduction of the reproductive output

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

              (n = 10)

Nominal Loading of the Saturated Solution [mg test item/L]	Adult Mortality [%]
	7 d	14 d	21 d
100	0	0	0
Control	0	0	0

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading of the Saturated Solution [mg test item/L]	Day of First Brood										First
	in Replicate No.										Appearance
	1	2	3	4	5	6	7	8	9	10	Mean Day
100	9	9	10	10	8	10	8	10	10	10	9.4
Control	9	9	9	10	8	10	10	8	10	10	9.3

Total Length and Dry Weight of the Parent Animals

Nominal Loading of the Saturated Solution [mg test item/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
100	5.50	5.50	5.50	5.50	5.75	5.50	5.25	5.75	5.50	5.25	5.50	10	9.1	0.91
Control	5.50	5.25	5.50	5.25	5.25	5.50	5.25	5.50	5.25	5.50	5.38	10	7.8	0.78

Validity criteria fulfilled:

yes

Conclusions:

In the saturated solution of the test item Tetrabutane (industrial) with a nominal loading concentration of 100 mg/L, no statistically or biologically significant effects were observed. The No Observed Effect Loading (NOEL) and the Lowest Observed Effect Loading (LOEL) were assessed at:

NOEL: 100 mg/L (saturated solution)
LOEL: > 100 mg/L (saturated solution)

All effects given are based on the nominal loading of the saturated solution of the test item Tetrabutane (industrial).

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the saturated solution* of the test item Tetrabutane (industrial) (batch number: 16.06.2008) was conducted as a limit test according to OECD 211 (2008) from 2009-11-10 to 2010-03-16, with the definitive exposure phase from 2010-02-22 to 2010-03-15, at Dr.U.Noack-Laboratorien in Sarstedt, Germany.
Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the saturated solution and control. At test start they were 2 to 24 h old. The test method was semi-static. The test solutions were renewed 3 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 on parameters such as the intrinsic rate of natural increase, adult mortality, the occurrence of aborted eggs and stillborn juveniles, time of production of first brood, body weight and length of the parent animals. The limit concentrationand the control were analytically verified via GC- FID of samples on days 0, 7, 18 (fresh media, 0 h) and on days 2, 9, 21 (old media, 48 h).

The recoveries of the test item were lower than the LOQ of 20 µg/L. The concentration of the saturated solution of the test item Tetrabutane (industrial) was not determinable. All effects given are based on the saturated solution of the test item Tetrabutane (industrial).

·   The average number of juveniles per parent in the control group was 111 and 107 in the saturated solution of the test item after 21 days. The reproductive output of the saturated solution was comparable to the control group and not statistically significant reduced (t-test, p = 0.05).

·   The coefficient of variation of the mean number of living offspring produced per parent in the control group was 12 % and 8 % in the saturated solution.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance (Table 1). The intrinsic rates of natural increase (IR) of the treatment groups were compared to the control by Mann-whitney Rank Sum Test (p = 0.05), because the normality test failed. The mean IR of the surviving daphnids of the saturated solution was comparable to the control group.

·   No immobilization or mortality of parent animals occurred in the control or test groups.

·   No stillborn juveniles and aborted eggs were produced by the control group and the saturated solution.

*)    Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6. 

·   The mean day of release of first brood was day 9.3 in the control and day 9.4 in the saturated solution. In the control as well as in the test groups the first brood occurred until day 10. In the control and the test groups four to five broods were released during the test period by all daphnids (Table 1).

·   No males and ephippia (winter eggs) occurred in control or test groups.

A summary of all test item related effects is given in Table 1.
There was no statistically or biologically significant effect observed in the saturated solution of the test item Tetrabutane (industrial), when compared to the control.

Table 1:    Summary of all Test Item Related Effects

              Based on the nominal loading of the test item Tetrabutane (industrial)

Effects	Tetrabutane (industrial) Nominal Loading of the Test Item [mg/L]		Significance
	Control	100
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	111 ± 13.2	107 ± 8.53	no^1)
Mean Intrinsic Rate of Natural Increase	0.44	0.44	no^2)
Appearance of First Brood [Mean Day]	9.3	9.4	no^3)
Mean Number of Broods	4.6	4.3	no^3)
Stillborn Juveniles and Aborted Eggs	0	0	no^3)
Adult Mortality after 21 days [%]	0	0	no^3)
NOELReproduction	100 mg/L (saturated solution)
LOELReproduction	> 100 mg/L (saturated solution)

SD    = Standard deviation

no     = No significance

¹⁾       = Statistically significant difference (t-test, p = 0.05)

²⁾       = Statistically significant difference (Mann-whitney Rank Sum Test, p = 0.05)

³⁾       = Biologically significant

 

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

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.60 mg/L after 24 h was 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.

Description of key information

Tetrabutan is not harmful to daphnids by  long-term exposure.
NOEL 100 mg/L (saturated solution) under the conditions of the test (OECD 211).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Tetrabutane (Industrial) was tested for long-term toxicity with daphnids (Daphnia magna Straus) under semi-static conditions according to the guideline OECD 211 (Daphnia magna reproduction test). The test was conducted as a limit test in a saturated solution of Tetrabutane with a nominal loading concentration of 100 mg/L. The achieved concentration of Tetrabutane in the saturated solution was not determined, as recoveries of the test substance were below the LOQ of 20 µg/l. No statistically or biologically significant effects were observed. The No Observed Effect Loading (NOEL) and the Lowest Observed Effect Loading (LOEL) were assessed as 100 mg/l (saturated solution) and > 100 mg/l (saturated solution), respectively .