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EC number: 6297151  CAS number: 1226892438
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 Ecotoxicological Summary
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 Toxicity to other aquatic organisms
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Sediment toxicity
Administrative data
Link to relevant study record(s)
 Endpoint:
 sediment toxicity: longterm
 Type of information:
 experimental study
 Adequacy of study:
 key study
 Study period:
 20200831 to 20201009
 Reliability:
 1 (reliable without restriction)
 Rationale for reliability incl. deficiencies:
 guideline study
 Qualifier:
 according to guideline
 Guideline:
 OECD Guideline 225 (SedimentWater Lumbriculus Toxicity Test Using Spiked Sediment)
 Version / remarks:
 2007
 Deviations:
 no
 GLP compliance:
 yes (incl. QA statement)
 Analytical monitoring:
 yes
 Details on sampling:
 The limit test item concentration and the controls were confirmed by analytical verification via LCMS/MS on day 2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each. Determination of the test item was performed for the sediment, pore water and the overlying water in the controls and limit test item concentration under GLP. Except for the samples of day 2 and 0 all replicates contained worms. The method was implemented under nonGLP and documented finally in the GLP raw data.
 Vehicle:
 yes
 Remarks:
 Acetone
 Details on sediment and application:
 Sediment
Artificial sediment containing:
 4 % peat, airdried and finely ground
 20 % kaolin, with a kaolinite content > 30 %
 76 % quartz sand (> 50 % particles sized 50200 µm)
 0.2 % calcium carbonate (CaCO3) to achieve a pH of 6.92
The organic carbon content of the final mixture was 2.35 % (external determination, nonGLP).
Application
The respective test item amount was weighed out for the limit test item concentration and dissolved in acetone.
The appropriate amount of spiking solution (2.5 mL per replicate) was thoroughly mixed with a subquantity of the artificial sediment (10 g per replicate). After complete evaporation of the solvent, the spiked portion of artificial sediment was thoroughly mixed
with the remaining sediment. Demineralised water was added to the artificial sediment to adjust the humidity of the artificial sediment to a moisture of 30 %.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 45 g soil dry weight were filled into the test vessels.
 Test organisms (species):
 Lumbriculus variegatus
 Details on test organisms:
 Test system Lumbriculus variegatus (Müller)
Lumbriculidae, Oligochaeta
Reason for the selection of the test system
Lumbriculus variegatus is a recommended species according to the guideline.
Origin
Bundesanstalt für Gewässerkunde (BfG), Am Mainzer Tor 1,
56068 Koblenz, Germany
Breeding
Breeding is performed at the test facility at 20 2 °C and diffuse light (100  500 lx, 16 h photoperiod daily).
The dissolved oxygen concentration is > 70 % of the air saturation value corresponding to 6.2 mg O2/L.
All worms used in the test originated from the same delivery of the supplier and the same stock. They were not used in another test.
Medium
Breeding of L. variegatus is performed on quartz sand in
reconstituted water containing 125 mL from each stock solution
poured in a can and filled up to 25 L with demineralised water.
Stock solution [g/L]
CaCl ∙ 2 H2O 55.80
MgSO4 ∙ 7 H2O 24.65
NaHCO3 12.95
KCl 1.15
Feeding TetraMin® flake food (TETRA) is provided twice per week.
The worms were synchronized 13 days prior to test start to minimize uncontrolled reproduction and regeneration.
At test start the worms had a body weight of 8.30 mg dry weight/10 worms (mean weight of two determinations).  Study type:
 laboratory study
 Test type:
 static
 Water media type:
 other: Same as for holding. Overlying Test Water Properties at Day 2 pHvalue Hardness [mgl/L CaCO3] Oxygen content [mg/L] Reconstituted Water used as Overlying Test Water 7.70 244 8.32
 Type of sediment:
 artificial sediment
 Remarks:
 Sediment Properties at Test Start Total Organic Carbon [%] 2.35* pHvalue 6.92 Moisture [%] Sediment at test start 30 *) external measurement (20200812 to 20200814), nonGLP
 Limit test:
 yes
 Duration:
 28 d
 Exposure phase:
 total exposure duration
 Remarks:
 20200902 to 20200930
 Hardness:
 Overlying Test Water Properties at Day 2
Hardness
[mgl/L CaCO3]
Reconstituted Water used as Overlying Test Water 244
Water Parameters ( Hardness)
Day Parameter Control Solvent Control Nominal test item concentration
1000 mg test item/kg sediment dry weight
0
(before
insertion of worms) Total hardness [mg/L CaCO3] 291 301 313
28 Total hardness [mg/L CaCO3] 298 291 285  Test temperature:
 Water Parameters (Temperature)
Day Parameter Control Solvent Control Nominal test item concentration
1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) Temperature [°C] 21.1 21.4 21.6
6 Temperature [°C] 20.0 19.8 19.8
13 Temperature [°C] 20.0 19.7 19.7
20 Temperature [°C] 19.8 19.2 19.3
28 Temperature [°C] 20.2 20.0 19.8  pH:
 Overlying Test Water Properties at Day 2
pHvalue
Reconstituted Water used as Overlying Test Water 7.70
Water Parameters (pHvalue)
Day Parameter Control Solvent Control Nominal test item concentration
1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) pH 7.59 7.57 7.53
6 pH 7.81 7.77 7.75
13 pH 7.82 7.80 7.76
20 pH 7.84 7.70 7.69
28 pH 7.39 7.50 7.58
 Dissolved oxygen:
 Overlying Test Water Properties at Day 2
Oxygen content
[mg/L]
Reconstituted Water used as Overlying Test Water 8.32
Water Parameters (OxygenConcentration)
Day Parameter Control Solvent Control Nominal test item concentration 1000 mg test item/kg sediment dry weight
0
(before
insertion of worms) O2 [mg/L]* 8.12 8.22 8.09
6 O2 [mg/L]* 8.65 8.45 8.51
13 O2 [mg/L]* 8.63 8.46 8.64
20 O2 [mg/L]* 8.35 8.61 8.69
28 O2 [mg/L]* 8.48 8.66 8.70
*) 70 % of the air saturation value corresponds to 6.2 mg O2/L.  Ammonia:
 AmmoniumN [mg/L]
Day after worm insertion Control Solvent Control mg test item/kg SDW
1000
0 0.35 < 0.05 0.14
2 0.11 0.53 0.49
5 < 0.05 0.09 0.33
7 0.52 0.30 1.55
9 0.42 0.43 1.54
12 0.09 0.36 1.17
14 0.15 0.09 0.68
16 0.21 0.15 0.37
19 0.26 0.14 1.16
21 0.48 0.15 1.07
23 0.35 0.16 0.92
26 0.34 0.51 1.32
28 0.43 0.23 1.06  Nominal and measured concentrations:
 Nominal test item concentration:
limit test item concentration with 1000 mg/kg sediment dry weight
The measured concentration of the test item in the sediment was 114% at day 0, 87% at day 7 and 47% at day 28. Minor recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ.  Details on test conditions:
 Environmental Conditions
Room temperature [°C] Photoperiod [h light] Light intensity [lx]
Nominal 20 ± 2 16 100  500
Actual 19 – 22 16 295 ± 56.7  Reference substance (positive control):
 yes
 Duration:
 28 d
 Dose descriptor:
 LOEC
 Effect conc.:
 > 1 000 mg/kg sediment dw
 Nominal / measured:
 nominal
 Conc. based on:
 act. ingr.
 Basis for effect:
 total number of worms
 Duration:
 28 d
 Dose descriptor:
 LOEC
 Effect conc.:
 > 1 000 mg/kg sediment dw
 Nominal / measured:
 nominal
 Conc. based on:
 act. ingr.
 Basis for effect:
 reproduction
 Duration:
 28 d
 Dose descriptor:
 LOEC
 Effect conc.:
 > 1 000 mg/kg sediment dw
 Nominal / measured:
 nominal
 Conc. based on:
 act. ingr.
 Basis for effect:
 biomass
 Key result
 Duration:
 28 d
 Dose descriptor:
 NOEC
 Effect conc.:
 > 1 000 mg/kg sediment dw
 Nominal / measured:
 nominal
 Conc. based on:
 act. ingr.
 Basis for effect:
 reproduction
 Remarks on result:
 other: 100% active ingredient content
 Details on results:
 After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to the pooled control in the limit test item concentration. All validity criteria were fulfilled.
Mortality / Reproduction of Worms
No mortality of worms was observed in the controls and the limit test item concentration after exposure to the test item over 28 days .
The total number of worms and thus the reproduction did not significantly differ compared to the pooled control in the limit test item concentration.
Behaviour and Pathological Symptoms
No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in the limit test item concentration during the 28 days of exposure.
Dry Biomass of the Worms per Replicate
At test start the worms had a body weight of 8.30 mg dry weight/10 worms (mean weight of two determinations). At test end, the mean biomass dry weight of the worms was not statistically significantly different at the limit test item concentration compared to the pooled control.  Results with reference substance (positive control):
 A reference item study is performed once a year with Potassium chloride. The most recent reference item test was carried out from 20200622 to 20200723 (StudyNo. 72006IRS/RLA70116). The study was conducted with four reference item concentrations of 1.0  2.0  4.0  8.0 g/kg sediment dry weight.
Summary of all Effects and resulting NOEC, LOEC and EC50values of the Reference Item Study
Effects Potassium Chloride
[g/kg sediment dry weight]
NOEC
Total worm number, Biomass 2
LOEC
Total worm number, Biomass 4
EC50 Total worm number
(CI 95%) 2.40 (1.99 – 2.86)
EC50 Biomass
(CI 95%) 2.61 (1.83 – 3.72)
CI = Confidence Interval  Reported statistics and error estimates:
 NOEC/LOECvalues and statistical analyses
Since no statistically significant differences were observed between the control and the solvent
control, both controls were pooled for further evaluation.
Total worm number  ShapiroWilk’s Test on Normal Distribution
(reproduction)  Levene’s Test on Variance Homogeneity (with Residuals)
 Twosample ttest Procedure
Biomass  ShapiroWilk’s Test on Normal Distribution
 Levene’s Test on Variance Homogeneity (with Residuals)
 Twosample ttest Procedure
The alphavalue (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.
Software Calculations and graphics were carried out using software:
 Excel, MICROSOFT CORPORATION
 ToxRat Professional, TOXRAT SOLUTIONS GMBH
 Sigma Plot, SPSS INC.
All data presented in the tables were computergenerated and have been rounded for presentation. Thus, manual calculations of results based on the data in this report may yield in minor deviations from these figures.  Validity criteria fulfilled:
 yes
 Conclusions:
 After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to the pooled control in the limit test item concentration.
All validity criteria were fulfilled.  Executive summary:
Summary
The effects of the test item FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE, Batch number 1865159) on the endobenthic oligochaete Lumbriculus variegatus in a watersediment system were determined at the test facility in Sarstedt, Germany, from 20200831 to 20201009, with the definitive exposure phase from 20200902 to 20200930. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the limit test item concentration 1000 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial soil treated with the solvent were set up. Six replicates per control and limit test item concentration were set up for biological investigations. Water quality parameters (temperature, pHvalue, O2concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentration, each.
Analytical evaluation of the test item FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE and controls was carried out via LCMS/MS on day 2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each.
The measured concentration of the freshly spiked artificial sediment on day 2 was 117 %, thus indicating its proper preparation. The control samples were < LOQ. After 7 and 28 days 87 and 47% were recovered respectively.
The pore water concentration in these samples remained relatively constant
After 28 days of exposure, FATTY ACIDS, C18 UNSAT, REACTION PRODUCTS WITH DIETHYLENETRIAMINE induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to pooled control in the limit test item concentration.
All validity criteria were fulfilled.
Summary of all Effects, Based on Nominal Test Item Concentration
Effects
Fatty acids, C18 unsat, reaction products with diethylenetriamine
[mg/kg sediment dry weight]
LOEC
(Total number of worms/Reproduction, Biomass)
>1000
NOEC
(Total number of worms/Reproduction, Biomass)
≥1000
Reference
Summary of all Effects, Based on Nominal Test Item Concentration
Effects 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 

[mg/kg sediment dry weight] 
LOEC (Total number of worms/Reproduction, Biomass) 
>1000 
NOEC (Total number of worms/Reproduction, Biomass) 
≥1000 
Mortality after 28 Days of Exposure
Nominal test item concentration [mg/kg sediment dry weight] 
Number of Dead or Missing 
MV 
SD ± 
Mortality[%] 

1 
2 
3 
4 
5 
6 

Control 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Solvent Control 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Pooled Control 
 
0 
0 
0 

1000 
0 
0 
0 
0 
0 
0 
0 
0 
0 
MV = Mean Value SD = Standard Deviation
Total Worm Number after 28 Days of Exposure
Nominal test item concentration [mg/kg sediment dry weight] 
Number of Surviving 
MV 
SD ± 
Inhibition to pooled control [%]^{#} 
Sign.* 

1 
2 
3 
4 
5 
6 

Control 
35 
24 
18 
22 
25 
38 
27.0 
7.80 
 
 

Solvent Control 
18 
36 
29 
37 
18 
24 
27.0 
8.44 
 
 

Pooled Control 
 
27.0 
7.75 
 
 

1000 
28 
23 
16 
28 
19 
15 
21.5 
5.75 
20.4 
No 

*) = Twosample ttest Procedure, alpha = 0.05
MV = Mean Value SD = Standard Deviation #) = Negative value = Increase compared to pooled control
Reproduction after 28 days of Exposure
Nominal test item concentration [mg/kg sediment dry weight] 
Number of Reproduced 
MV 
SD ± 
Inhibition to pooled control [%]^{#} 
Sign.* 

1 
2 
3 
4 
5 
6 

Control 
25 
14 
8 
12 
15 
28 
17.0 
7.80 
 
 

Solvent Control 
8 
26 
19 
27 
8 
14 
17.0 
8.44 
 
 

Pooled Control 
 
17.0 
7.75 
 
 

1000 
18 
13 
6 
18 
9 
5 
11.5 
5.75 
32.4 
N 
MV = Mean Value SD = Standard Deviation *) = ttest, alpha = 0.05
Worm Behaviour during 28 Days of Exposure
Nominal test item concentration 
Effect 
Days after addition 

0 
2 
5 
7 
9 
12 
14 
16 
19 
21 
23 
26 
28 



Number of replicates (out of 6 or 4 replicates) showing particular feature 

Control 
ob 
6 
4 
5 
6 
4 
5 
4 
6 
6 
6 
5 
4 
5 
a k 
  
2  
1  
  
2  
1  
2  
  
  
  
1 1 
2  
1  

f 
 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 

Solvent Control 
ob 
6 
5 
5 
6 
6 
4 
3 
5 
4 
6 
5 
6 
4 
a 
 
1 
1 
 
 
2 
3 
1 
2 
 
1 
 
2 

f 
 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 

1000 
ob a k f 
6    
4 2  6 
5 1  6 
6   6 
4 2  6 
4 2 1 6 
4 2 1 6 
4 2  6 
4 2  6 
4 2  6 
3 3  6 
6   6 
6   6 
a) worm on sediment surface ob) no abnormality detected f) faeces on the sediment surface
k) worms stick together ) no observation
Worm Dry Biomass after 28 Days of Exposure
Nominal test item concentration 
Dry weight[mg] per replicate 
MV 
SD ± 
Inhibition to control[%]^{#} 
Sign.* 

1 
2 
3 
4 
5 
6 

Control 
27.7 
19.5 
8.4 
14.1 
18.9 
39.0 
21.3 
10.8 
 
 

Solvent Control 
9.7 
30.0 
26.5 
37.6 
9.7 
10.0 
20.6 
12.4 
 
 

Pooled Control 
 
20.9 
11.1 
 
 

1000 
23.9 
19.9 
11.1 
11.3 
10.3 
10.2 
14.5 
5.9 
31.0 
N 
MV = Mean value SD = Standard deviation
*) Statistically significantly different compared to pooled control (Twosample ttest Procedure)
Statistical Analyses
Threshold concentrations (NOEC) for Worm Number at 28 d
Comparison between Control and Solvent Control for Worm Number at 28 d
ShapiroWilk´s Test on Normal Distribution
ShapiroWilk´s Test on Normal Distribution with worm number at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [mg/kg] Mean s n
Control 27.0 7.80 6
Solvent Control 27.0 8.44 6
Results:
Number of residuals = 14; ShapiroWilk´s W = 0.923; p(W) = 0.241; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (ShapiroWilk´s; p > 0.01).
Variance homogeneity check (Ftest) was passed (p > 0.01).
STUDENTt test for Homogeneous Variances
STUDENTt test for Homogeneous Variances with worm number at 28 d: Twosample comparison of the two controls. Significance was Alpha = 0.050, twosided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): twosided probability of F computed by the Ftest (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).
Treatm. [mg/kg] Mean s df %MDD t p(t) Sign. p(F)
Control 27.0 7.80
Solvent Control 27.0 8.44 10 38.7 0.00 1.000  0.867
+: significant; : nonsignificant
There is no statistically significant difference between control and solvent control.
Statistical Characteristics of the Samples
Statistical characteristics with worm number at 28 d: Mean: arithmetic mean (X); Med: median; Min: minimum value. Max: maximum value; n: sample size; s: standard deviation; s%: coefficient of variation; s(X): standard error; %s(X): %standard error; 95%l. 95%u: lower. upper 95%confidence limits.
Treatm. [mg/kg] Mean Med Min Max n s %s s(X) %s(X) 95%l 95%u
Pooled Control 27.0 24.5 18.0 38.0 12 7.75 28.7 2.24 8.3 22.1 31.9
1000.000 21.5 21.0 15.0 28.0 6 5.75 26.8 2.35 10.9 15.5 27.5
ShapiroWilk´s Test on Normal Distribution
ShapiroWilk´s Test on Normal Distribution with worm number at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.
Treatm. [mg/kg] Mean s n
Pooled Control 27.0 7.75 12
1000.000 21.5 5.75 6
Results:
Number of residuals = 14; ShapiroWilk´s W = 0.923; p(W) = 0.241; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with worm number at 28 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 13.444 1 13.444 1.415 0.252
Residuals 152.000 16 9.500
Total 165.44 17
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normaldistribution and variancehomogeneity requirements are fulfilled.
A parametric multiple test is advisable.
Twosample ttest Procedure
Twosample ttest Procedure with worm number at 28 d: Twosample comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, onesided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha (The residual variance of an ANOVA was applied; df = N  k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [mg/kg] Mean s df %MDD t p(t) Sign.
Pooled Control 27.0 7.18
1000.000 21.5 7.18 16 23.2 1.53 0.073  +: significant; : nonsignificant
There is no statistically significant difference between Pooled Control and 1000.000 mg/kg.
Threshold concentrations (NOEC) for Biomass after 28 days
Comparison between Control and Solvent Control for Biomass after 28 days
ShapiroWilk´s Test on Normal Distribution
ShapiroWilk´s Test on Normal Distribution with weight at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.
Treatm. [mg/kg] Mean s n
Control 21.2667 10.78419 6
Solvent Control 20.5833 12.34624 6
Results:
Number of residuals = 17; ShapiroWilk´s W = 0.938; p(W) = 0.292; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (ShapiroWilk´s; p > 0.01).
Variance homogeneity ckeck (Ftest) was passed (p > 0.01).
STUDENTt test for Homogeneous Variances
STUDENTt test for Homogeneous Variances with weight at 28 d: Twosample comparison of the two controls. Significance was Alpha = 0.050, twosided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): twosided probability of F computed by the Ftest (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).
Treatm. [mg/kg] Mean s df %MDD t p(t) Sign. p(F)
Control 21.2667 10.78419
Solvent Control 20.5833 12.34624 10 70.1 0.10 0.921  0.774
+: significant; : nonsignificant
There is no statistically significant difference between control and solvent control.
ShapiroWilk´s Test on Normal Distribution
ShapiroWilk´s Test on Normal Distribution with weight at 28 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.
Treatm. [mg/kg] Mean s n
Pooled Control 20.9250 11.05787 12
1000.000 14.4500 5.92343 6
Results:
Number of residuals = 17; ShapiroWilk´s W = 0.941; p(W) = 0.330; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with weight at 28 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 77.293410 1 77.293410 3.858 0.067
Residuals 320.593936 16 20.037121
Total 397.88735 17
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normaldistribution and variancehomogeneity requirements are fulfilled.
A parametric multiple test is advisable.
Twosample ttest Procedure
Twosample ttest Procedure with weight at 28 d: Twosample comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. onesided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha (The residual variance of an ANOVA was applied; df = N  k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [mg/kg] Mean s df %MDD t p(t) Sign.
Pooled Control 20.9250 9.74833
1000.000 14.4500 9.74833 16 40.7 1.33 0.101 
+: significant; : nonsignificant
There is no statistically significant difference between Pooled Control and 1000.000 mg/kg.
Peak areas related to measured concentrations
Measured peak areas and related concentrations may be different, because of daily calibration analysis
Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Artificial sediment and Sediment)
Nominal test item concetration [mg/kg SDW] 
1000 

Sampling date 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 

Absolute Response 
Calc. amount [µg/L] 
Dilution factor 

Inj. 1 
Inj. 2 
Inj. 3 
Inj. 1 
Inj. 2 
Inj. 3 

2 
3573.974 
3438.882 
 
5.987 
5.758 
 
20000 
0 
2578.629 
 
 
4.423 
 
 
20000 
7 
1339.793 
1353.962 
 
3.479 
3.511 
 
20000 
28 
1141.606 
1868.273 
1804.823 
2.589 
4.124 
3.990 
10000 
Calc.. amount Calculated amount of the test item without weighing and dilution factors
Inj. Injection
 Not determined
Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Aqueous Layer)
Nominal test item concetration [mg/kg SDW] 
1000 

Sampling date 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 

Absolute Response 
Calc. amount [µg/L] 
Dilution factor 

Inj. 1 
Inj. 2 
Inj. 1 
Inj. 2 

0 
7187.447 
6663.283 
0.959 
0.910 
20 
7 
28760.076 
27825.215 
4.514 
4.371 
20 
28 
5347.490 
6428.223 
0.874 
1.111 
20 
Calc.. amount Calculated amount of the test item without weighing and dilution factors
Inj. Injection
Calculated Amounts, Dilution factors and Responses of Fatty acids, C18 unsat, reaction products with diethylenetriamine (Pore Water)
Nominal test item concetration [mg/kg SDW] 
1000 

Sampling date 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 

Absolute Response 
Calc. amount [µg/L] 
Dilution factor 

Inj. 1 
Inj. 2 
Inj. 1 
Inj. 2 

0 
48551.934 
50726.215 
7.538 
7.870 
20 
7 
42772.543 
41741.641 
6.655 
6.497 
200 
28 
34391.086 
35444.617 
7.222 
7.452 
200 
Calc.. amount Calculated amount of the test item without weighing and dilution factors
Inj. Injection
Measured Concentrations and Percent of Nominal of Fatty acids, C18 unsat, reaction products with diethylenetriamine (artificial sediment) for Day 2
Sampling date 
Day 2 

Artificial sediment used for preparation of the test item concentration [mg/kg] 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 

Meas. conc. [mg/kg] 
% 

1000 
1170 
117 
Solvent Control 
< LOQ 

Control 
< LOQ 
Meas. conc. = Measured concentration (dilution factor taken into account)
% = Percent of the nominal concentration of the test item of the quartz sand
LOQ = 100 mg/kg of the test item
Measured Concentrations and Recovery Rates of Test Item (Aqueous Layer, Pore Water and Sediment)  Day 0
Nominal test item concentration [mg/kg SDW] 
1000 
Control 
Solvent Control 

Amount applied per test vessel [mg]^{1)} 
45.0 

Aqueous Layer 
Meas. amount 
< LOQ 
< LOQ 
< LOQ 
% 

Pore 
Meas. amount 
0.000585 
< LOQ 
< LOQ 
% 
0 

Sediment 
Meas. amount 
51.5 
< LOQ 
< LOQ 
% 
114 

Total 
[mg] 
51.5 
< LOQ 
< LOQ 
1) = Based on 45 g sediment (DW) and 150 mL dilution water
Meas. amount = Measured amount, dilution and weighing factor taken into account
% = Percent of the nominal concentration of the test item
LOQ = Limit of quantification of the analytical method (sediment: 100 mg test item/kg, aqueous layer: 100 µg test item/L)
Measured Concentrations and Recovery Rates of Test Item (Aqueous Layer, Pore Water and Sediment)  Day 7 and 28
Day 7 

Nominal test item concentration [mg/kg SDW] 
1000 
Control 
Solvent Control 

Amount applied per test vessel [mg]^{1)} 
45.0 

Aqueous Layer 
Meas. amount 
0.130 
< LOQ 
< LOQ 
% 
0 

Pore 
Meas. amount 
0.00516 
< LOQ 
< LOQ 
% 
0 

Sediment 
Meas. amount 
39.2 
< LOQ 
< LOQ 
% 
87 

Total 
[mg] 
39.3 
< LOQ 
< LOQ 
% 
87 

Day 28 

Nominal test item concentration [mg/kg SDW] 
1000 
Control 
Solvent Control 

Amount applied per test vessel [mg]^{1)} 
45.0 

Aqueous Layer 
Meas. amount 
0.00330 
< LOQ 
< LOQ 
% 
0 

Pore 
Meas. amount 
0.00629 
< LOQ 
< LOQ 
% 
0 

Sediment 
Meas. amount 
21.3 
< LOQ 
< LOQ 
% 
47 

Total 
[mg] 
21.3 
< LOQ 
< LOQ 
1) = Based on 45 g sediment (DW) and 150 mL dilution water
Meas. amount = Measured amount, dilution and weighing factor taken into account
% = Percent of the nominal concentration of the test item
LOQ = Limit of quantification of the analytical method (sediment: 100 mg test item/kg, aqueous layer: 100 µg test item/L)
Description of key information
There is one long term sediment test with lumbriculus variegatus available which was performed according to OECD 225 under GLP conditions and fulfilled all validity criteria.
The test was performed as limit test at 1000 mg a.i./kg dw and no effects were observed.
In addition to measured data the PNECsed was calculated using the equilibrium partitioning method (EPM).
Key value for chemical safety assessment
 EC10, LC10 or NOEC for freshwater sediment:
 1 000 mg/kg sediment dw
Additional information
The effects of Fatty acids, C18 unsat, reaction products with diethylenetriamine (AAIDETA) on the endobenthic oligochaete Lumbriculus variegatus in a watersediment system were determined under GLP conditions. The study was formed according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the limit test item concentration 1000 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial sediment treated with the solvent were set up. Six replicates per control and limit test item concentration were set up for biological investigations. Water quality parameters (temperature, pHvalue, O_{2}concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentration, each.
Analytical evaluation of the test item Fatty acids, C18 unsat, reaction products with diethylenetriamine and controls was carried out via LCMS/MS on day 2 from the freshly spiked artificial sediment and on day 0, 7 and 28 using additional replicates, each. The measured concentration of the freshly spiked artificial sediment on day 2 was 117 %, thus indicating its proper preparation. The control samples were < LOQ.
After 28 days of exposure, Fatty acids, C18 unsat, reaction products with diethylenetriamine induced no mortality in the limit test item concentration 1000 mg/kg sediment dry weight.
The total number of worms and thus the reproduction as well as the total biomass per replicate did not statistically significantly differ compared to pooled control in the limit test item concentration. For further endpoints, please see Table 1 below.
All validity criteria were fulfilled.
Table 1: Summary of all Effects, Based on Nominal Test Item Concentration
Effects 
Fatty acids, C18 unsat, reaction products with diethylenetriamine 
 [mg/kg sediment dry weight] 
LOEC (Total number of worms/Reproduction, Biomass)  > 1000 
NOEC (Total number of worms/Reproduction, Biomass)  ≥ 1000 
The PNECsed of 10 mg/kg dw is according to REACH guidance R.10 calculated from the available longterm lumbriculus test by applying the default safety factor of 100.
In the absence of any measured toxicity to sediment organisms the PNECsed can also be calculated using the Equilibrium Partitioning Method (EPM). Because of the availability of a longterm sediment test for a substance for which it is indicated that the use of EPM to predict the PNECsediment is not appropriate (surfactant, ionic). The PNECsed of 10 mg/kg dw will be used for risk assessment.
For comparison reasons the PNECsed was also calculated using the Equilibrium partitioning method (EPM). According to REACH guidance R.10 this method uses the PNEC_{aquatic }and the sediment/water partitioning coefficient as inputs.
The dose response results for the algae test are calculated based on nominal Test Loadings. The PNECaquatic is based on the Algae E_{r}L_{10} after 72 hours is 20.7 µg/L. The EL10 for daphnia was observed to be 133 µg/L.
The PNEC_{sed }is then calculated using the equations detailed in the TGD:
PNEC_{sed} = K_{suspwater}* PNEC_{aquatic }* 1000 * 1 / RHO_{susp}
Where: PNEC_{aquatic } = 2.07 µg/L
K_{suspwater} = 23500.9 m^{3}.m^{3}
RHO_{susp} = 1150 kg.m^{3}(TGD, equ. 18)
PNEC_{sedEPM} = 42.3 mg/kg ww = 194.6 mg/kg dw
Applying the additional safety factor for exposure via ingestion: 10
PNEC_{sedEPM} = 4.23 mg/kg ww
= 19.46 mg/kg dw
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